Treatment of hyperproliferative diseases with vinca alkaloid N-oxide and analogs

ABSTRACT

The present invention relates to vinca alkaloid and analog N-oxides having activity for treating hyperproliferative disorders. Further, the invention relates to pharmaceutical compositions and methods of using vinca alkaloid and analog N-oxides, alone or in combination with one or more other active agents or treatments, to treat hyperproliferative disorders.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a Continuation-in-part of PCT Application No.PCT/US2007/004252, filed Feb. 20, 2007. PCT Application No.PCT/US2007/004252 claims the benefit of the filing date of ProvisionalPatent Application No. 60/774,204, filed Feb. 17, 2006. The disclosureof each priority application is incorporated herein by reference in itsentirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to vinca alkaloid N-oxides having activityfor treating hyperproliferative disorders. Further, the inventionrelates to pharmaceutical compositions and methods of using vincaalkaloid N-oxides, alone or in combination with one or more other activeagents or treatments, to treat hyperproliferative disorders.

2. Related Art

One in every four deaths in the United States is due to cancer, andcancer is the second leading cause of death. U.S. Cancer StatisticsWorking Group; United States Cancer Statistics: 2000 Incidence, Atlanta(Ga.): Department of Health and Human Services, Centers for DiseaseControl and Prevention, and National Cancer Institute (2003). TheNational Cancer Institute reports that almost 10 million Americans havea history of invasive cancer, while the American Cancer Societyestimates that in the year 2004, over 1.3 million Americans will receivea diagnosis of invasive cancer with over a half million cases resultingin death. American Cancer Society, Cancer Facts & Figures 2004. Thesestatistics exclude the 1 million cases of basal and squamous cell skincancers that are expected to be diagnosed in the United States.

Cancers are classified based on the organ and cell tissue from which thecancer originates, including: (i) carcinomas (most common kind of cancerwhich originates in epithelial tissues, the layers of cells covering thebody's surface or lining internal organs and various glands); (ii)leukemias (origination in the blood-forming tissues, including bonemarrow, lymph nodes and the spleen); (iii) lymphomas (originates in thecells of the lymph system); (iv) melanomas (originates in the pigmentcells located among the epithelial cells of the skin); and (v) sarcomas(originates in the connective tissues of the body, such as bones,muscles and blood vessels). (See Molecular Biology of the Cell: ThirdEdition, “Cancer,” Chapter 24, pp. 1255-1294, B. Alberts et al., (eds.),Garland Publishing, Inc., New York (1994); and Stedman's Pocket MedicalDictionary; Williams and Wilkins, Baltimore (1987)). Within these broadcancer classifications, there are over one hundred cancersubclassifications, such as breast, lung, pancreatic, colon, andprostate cancer, to name a few.

Cancer cells develop as a result of damage to a cell's DNA (i.e.,altered DNA sequence or altered expression pattern) from exposure tovarious chemical agents, radiation, viruses, or when somenot-yet-fully-understood internal, cellular signaling event occurs. Mostof the time when a cell's DNA becomes damaged, the cell either dies oris able to repair the DNA. However, for cancer cells, the damaged DNA isnot repaired and the cell continues to divide, exhibiting modified cellphysiology and function.

Neoplasms, or tumors, are masses of cells that result from an aberrant,accelerated rate of growth (i.e., hyperproliferative cell growth). Aslong as the tumor cells remain confined to a single mass, the tumor isconsidered to be benign. However, a cancerous tumor has the ability toinvade other tissues and is termed malignant. In general, cancer cellsare defined by two heritable properties: the cells and their progeny 1)reproduce in defiance of normal restraints, and 2) invade and colonizethe territories of other cells.

Cancerous tumors are comprised of a highly complex vasculature anddifferentiated tissue. A large majority of cancerous tumors have hypoxiccomponents, which are relatively resistant to standard anti-cancertreatment, including radiotherapy and chemotherapy. Brown, Cancer Res.59:5863 (1999); and Kunz, M. et al., Mol. Cancer. 2:1 (2003). Thomlinsonand Gray presented the first anatomical model of a human tumor thatdescribes a 100 to 150 μm thick hypoxic layer of tissue located betweenthe blood vessels and necrotic tumor tissues.

Research has shown that the hypoxic tissues within a number of canceroustumors promote the progression of the cancer by an array of complexmechanisms. See, Brown., supra, and Kunz et al., supra. Among these areactivation of certain signal transduction pathways and gene regulatorymechanisms, induction of selection processes for gene mutations, tumorcell apoptosis and tumor angiogenesis. Most of these mechanismscontribute to tumor progression. Therefore, tissue hypoxia has beenregarded as a central factor for tumor aggressiveness and metastasis.Therapies that target hypoxic tissues within a tumor would certainlyprovide improved treatments to patients suffering from tumor-relatedcancers and/or disorders.

In addition to cancer, there exist a number of hyperproliferativediseases and/or disorders that are associated with the onset of hypoxiain a given tissue. For example, Shweiki et al. explains that inadequateoxygen levels often lead to neovascularization in order to compensatefor the needs of the hypoxic tissue. Neovascularization is mediated byexpression of certain growth factors, such as vascular endothelialgrowth factor (VEGF). Shweiki et al., Nature 359:843 (1992). However,when certain tissues or growth factors are either directly or indirectlyupregulated in response to hypoxia without sufficient feedbackmechanisms for controlling tissue expression, various diseases and/ordisorders may ensue (i.e., by hypoxia-aggravated hyperproliferation). Byway of example, hypoxia-aggravated hyperproliferative diseases and/ordisorders having over-expressed levels of VEGF include ocular angiogenicdiseases, such as age-related macular degeneration and diabeticretinopathy, as well as cirrhosis of the liver. See Frank, OphthalmicRes. 29:341 (1997); Ishibashi et al., Graefe's Archive Clin. Exp.Ophthamol. 235:159 (1997); Corpechot et al., Hepatology 35:1010 (2002).

Vinca alkaloids are a class of chemotherapeutic agents originallydiscovered in the Madagascar periwinkle. Currently known vinca alkaloidsinclude vinblastine, vincristine, vindesine and vinorelbine. Vincaalkaloids act by inhibiting mitosis in metaphase. These alkaloids bindto tubulin, thus preventing the cell from making the spindles it needsto be able to move its chromosomes around as it divides. These alkaloidsalso seem to interfere with cells' ability to synthesize DNA and RNA.They are all administered intravenously in their sulfate form once aweek; these solutions are fatal if they are administered incorrectly,and can cause considerable tissue irritation if they leak out of thevein. See U.S. Pat. No. 6,555,547 for further detail.

U.S. Pat. No. 6,365,735 discloses a process for preparing vincaalkaloids of the general formula (I):

in which:R′₁ represents a hydrogen atom or an alkoxy, acyl, formyl orhalogenoacyl group,R′₂ represents a hydrogen atom or an alkyl group,R′₃ and R″₃ are identical or different and each independently representsa hydrogen atom or a hydroxyl or alkanoyloxyl group, or else R′₃ and R″₃together form a carbonyl group or else R′₃ and R′₅ together form anepoxy bridge or a double bond,R′₄ represents a hydrogen atom or an alkyloxycarbonyl, hydroxymethyl oralkanoyloxymethyl group, preferably an alkyloxycarbonyl group,R′₅ and R″₅ are identical or different and each independently representsa hydrogen atom or a hydroxyl, alkanoyloxyl, ethyl or 2-hydroxyethylgroup,R′₆ represents a hydrogen atom or an ethyl, 2-hydroxyethyl or acetylgroup,R′₇ represents a hydrogen atom or a cyanide group,R₁ represents a hydrogen atom or an alkyl, formyl or acyl group,preferably hydrogen or an alkyl group,R₂ represents a hydrogen atom or an alkoxy group,R₃ represents a hydrogen atom or a hydroxyl or alkanoyloxyl group, orelse R₃ and R₄ together form an epoxy bridge or a double bond,R₄ represents a hydrogen atom or a hydroxyl or alkanoyloxyl group, orelse R₄ and R₅ together form an epoxy bridge,R₆ represents an alkyloxycarbonyl, hydrazido, acetamido, hydroxymethylor alkanoyloxymethyl group,R₅ and R₇ represent a hydrogen atom or a hydroxyl or alkanoyloxyl group,as well as their addition salts with acids and their quaternary ammoniumsalts.

Disclosed also are additional classes of vinca alkaloids as wellspecific vinca alkaloids such as vinblastine, vincristine,anhydrovinblastine and vinorelbine.

BRIEF SUMMARY OF THE INVENTION

The present invention is related to compounds, compositions and methodsfor treating hyperproliferative disorders, such as cancer andinflammation. One aspect of the invention is drawn to vinca alkaloidN-oxides having Formula I:

or a pharmaceutically acceptable salt thereof, wherein:

R′₁ represents hydrogen, alkyl, alkoxy, acyl, formyl or halogenoacylgroup;

R′₂ represents hydrogen or alkyl group;

R′₃ and R″₃ are identical or different and each independently representshydrogen, hydroxy or alkanoyloxy group, or else R′₃ and R″₃ togetherform a carbonyl group or else R′₃ and R′₅ together form an epoxy bridgeor a double bond;

R′₄ represents hydrogen, alkyloxycarbonyl, hydroxymethyl oralkanoyloxymethyl group, preferably an alkyloxycarbonyl group;

R′₅ and R″₅ are identical or different and each independently representshydrogen, hydroxy, alkoxycarbonyl, C₁-C₇ alkyl optionally substitutedwith 1-3 halogen atoms, alkanoyloxy, or 2-hydroxyethyl group;

R′₆ represents hydrogen, ethyl, 2-hydroxyethyl or acetyl group;

R′₇ represents hydrogen or cyano;

R₁ represents hydrogen, alkyl, formyl or acyl group, preferably hydrogenor an alkyl group,

R₂ represents hydrogen or alkoxy;

R₃ represents hydrogen, hydroxy or alkanoyloxy group, or else R₃ and R₄together form an epoxy bridge or a double bond;

R₄ represents hydrogen, hydroxy or alkanoyloxy group, or else R₄ and R₅together form an epoxy bridge;

R₆ represents an alkyloxycarbonyl, hydrazido, acetamido, hydroxymethyl,alkanoyloxymethyl group or —C(═O)-A-NH—P, where -A- is one of —NH—,—NH-alk-COO— or —NH-alk-COONH—, alk is a straight chain or branchedC₁-C₇ alkyl group and —NH—P is a peptide residue, or R₅ and R₆ togetherform an oxazolidine-2,4-dione ring;

R₅ and R₇ represent hydrogen, hydroxy or alkanoyloxy group, as well astheir addition salts with acids and their quaternary ammonium salts;

each of R₈ and R′₈ is O or is absent provided that at least one of R₈and R′₈ is O; and

n is 1 or 2.

In one embodiment, when n=2 and one of R′₅ and R″₅ forms a double bondtogether with R′₃ or R″₃, then the other is not ethyl.

In one embodiment, the compound having formula I is vinblastine N-oxide,desacetyl vinblastine N-oxide, vinorelbine N-oxide, vincristine N-oxideor vinflunine N-oxide, or a pharmaceutically acceptable salt thereof.

Another aspect of the present invention is related to methods fortreating hyperproliferative disorders. In certain aspects of theinvention, the hyperproliferative disorder is cancer. In one embodiment,the cancer is a solid tumor. In another embodiment, the cancer isselected from the group consisting of colon cancer, brain cancer,glioma, multiple myeloma, head and neck cancer, hepatocellular cancer,melanoma, ovarian cancer, cervical cancer, renal cancer, and non-smallcell lung cancer. In a further embodiment, the cancer is acute andchronic lymphocytic leukemia, acute granulocytic leukemia, adrenalcortex carcinoma, bladder carcinoma, breast carcinoma, cervicalcarcinoma, cervical hyperplasia, choriocarcinoma, chronic granulocyticleukemia, chronic lymphocytic leukemia, colon carcinoma, endometrialcarcinoma, esophageal carcinoma, essential thrombocytosis, genitourinarycarcinoma, hairy cell leukemia, head and neck carcinoma, Hodgkin'sdisease, Kaposi's sarcoma, lung carcinoma, lymphoma, malignant carcinoidcarcinoma, malignant hypercalcemia, malignant melanoma, malignantpancreatic insulinoma, medullary thyroid carcinoma, melanoma, multiplemyeloma, mycosis fungoides, myeloid and lymphocytic leukemia,neuroblastoma, non-Hodgkin's lymphoma, osteogenic sarcoma, ovariancarcinoma, pancreatic carcinoma, polycythemia vera, primary braincarcinoma, primary macroglobulinemia, prostatic carcinoma, renal cellcarcinoma, rhabdomyosarcoma, skin cancer, small-cell lung carcinoma,soft-tissue sarcoma, squamous cell carcinoma, stomach carcinoma,testicular carcinoma, thyroid carcinoma, or Wilms' tumor.

In further aspects of the invention the hyperproliferative disorder isany one of age-related macular degeneration, Crohn's disease, cirrhosis,chronic inflammatory-related disorders, proliferative diabeticretinopathy, proliferative vitreoretinopathy, retinopathy ofprematurity, granulomatosis, immune hyperproliferation associated withorgan or tissue transplantation, an immunoproliferative disease ordisorder, e.g., inflammatory bowel disease, psoriasis, rheumatoidarthritis, systemic lupus erythematosus (SLE), vascularhyperproliferation secondary to retinal hypoxia, or vasculitis.

In one embodiment the invention is drawn to methods of treating,ameliorating, or preventing hyperproliferative disease in a subjectcomprising administering to said subject a therapeutically effectiveamount of an N-oxide of vinca alkaloid or analog thereof. In anotherembodiment, the vinca alkaloid analog is selected from the groupconsisting of vinblastine N-oxide, desacetyl vinblastine N-oxide,vinorelbine N-oxide, vincristine N-oxide and vinflunine N-oxide, or apharmaceutically acceptable salt thereof.

In certain embodiments, a metronomic dosing regime for an N-oxide ofvinca alkaloid or analog thereof comprises administration of the N-oxideat a dose below an established maximum tolerated dose (MTD) for theN-oxide, which upon repeated administration inhibits tumor growth andproduces less toxic side effects as compared to administration of themaximum tolerated dose of the N-oxide. While not being bound to aparticular mechanism, it is believed that metronomic dosing with anN-oxide of vinca alkaloid or analog thereof may target cells of thevasculature which form the blood vessels of the tumor as opposed to thetumor cells themselves. Accordingly, inhibition of tumor growth mayresult from the inability of the tumor cells to establish the functionalmicrovasculature critical for tumor growth and dissemination.

An additional aspect of the present invention is a method for treating,ameliorating, or preventing hyperproliferative disorders in an animalcomprising administering to the animal a therapeutically effectiveamount of a compound having Formula I in combination with one or moreactive agents or treatments. In one embodiment, the one or more activeagent or treatment is a chemotherapeutic agent, a radiotherapeuticagent/treatment, an anti-angiogenesis agent, a vascular targeting agent,a hypoxia-inducible factor 1 (HIF1) inhibitor, an Hsp90 inhibitor, atyrosine kinase inhibitor, a serine/threonine kinase inhibitor, aproteasome inhibitor, an HDAC inhibitor, a caspase inducer, a CDKinhibitor, and a proapoptotic molecule. In another embodiment, the oneor more active agent or treatment is used, has been used, or is known tobe useful for the treatment of the hyperproliferative disorder.

A particular aspect of the present invention is a method for treatinghyperproliferative disorders in an animal comprising administering tothe animal a pharmaceutically acceptable amount of an N-oxide of vincaalkaloid or analog thereof, in combination with one or more othertherapeutic agents, including topoisomerase 1 inhibitors. In otherembodiments, topoisomerase 1 inhibitor can be any topoisomerase 1inhibitor which is used, has been used, or is known to be useful for thetreatment of hyperproliferative disorders. Examples of topoisomerase 1inhibitors include camptothecin and its analogs such as those describedin U.S. Pat. No. 6,350,756 (for example,9-dimethylaminomethyl-10-hydroxycamptothecin (topotecan),7-ethyl-10[4-(1-piperidino)-1-piperidino]carbonyloxycamptothecin(irinotecan), 9-aminocamptothecin, 10-aminocamptothecin,10,11-methylenedioxycamptothecin and 7-ethyl-10-hyrdoxycamptothecin(SN-38)). In certain embodiments, the vinca alkaloid analog N-oxide usedin the combination therapy is selected from the group consisting ofvinblastine N-oxide, desacetyl vinblastine N-oxide, vinorelbine N-oxide,vincristine N-oxide and vinflunine N-oxide, or a pharmaceuticallyacceptable salt thereof. In other embodiments, the compound of Formula Iis administered 1 mg/kg q3d×5 to 500 mg/kg q3d×5. In other embodiments,the compound of Formula I is administered 5 mg/kg q3d×5 to 50 mg/kgq3d×5.

In other embodiments, irinotecan, or a pharmaceutically acceptable saltthereof, is administered 50 mg/m² to 500 mg/m² once weekly, once everytwo weeks or once every three weeks.

In one embodiment, the method of treating, ameliorating, or preventinghyperproliferative disorder in an animal comprises administering to theanimal a therapeutically effective amount of vinca alkaloid N-oxide oranalog thereof. In particular embodiments, the vinca alkaloid analogN-oxide is selected from the group consisting of vinblastine N-oxide,desacetyl vinblastine N-oxide, vinorelbine N-oxide, vincristine N-oxideand vinflunine N-oxide, or a pharmaceutically acceptable salt thereof,in combination with one or more active agents or treatments, forexample, chemotherapeutic agents or radiotherapeutic agents/treatments.

In preferred embodiments of the invention, the one or morechemotherapeutic agents can be any chemotherapeutic agent which is used,has been used, or is known to be useful for the treatment ofhyperproliferative disorders.

In preferred embodiments of the invention, the one or moreradiotherapeutic agents or treatments can be external-beam radiationtherapy, brachytherapy, thermotherapy, radiosurgery, charged-particleradiotherapy, neutron radiotherapy, photodynamic therapy, orradionuclide therapy.

In one embodiment of the invention, the compound having Formula I can beadministered prior to, during, and/or beyond administration of the oneor more chemotherapeutic agents or radiotherapeutic agents ortreatments. In another embodiment of the invention, the method ofadministering a compound having Formula I in combination with one ormore chemotherapeutic agents or radiotherapeutic agents or treatments isrepeated more than once.

The combination of a compound having Formula I and one or morechemotherapeutic agents or radiotherapeutic agents or treatments of thepresent invention will have additive potency or an additive therapeuticeffect. The invention also encompasses synergistic combinations wherethe therapeutic efficacy is greater than additive. Preferably, suchcombinations will reduce or avoid unwanted or adverse effects. Incertain embodiments, the combination therapies encompassed by theinvention will provide an improved overall therapy relative toadministration of a compound having Formula I or any chemotherapeuticagent or radiotherapeutic agent or treatment alone. In certainembodiments, doses of existing or experimental chemotherapeutic agentsor radiotherapeutic agents or treatments will be reduced or administeredless frequently which will increase patient compliance, therebyimproving therapy and reducing unwanted or adverse effects.

Further, the methods of the invention will be useful not only withpreviously untreated patients but also will be useful in the treatmentof patients partially or completely refractory to current standardand/or experimental cancer therapies, including but not limited toradiotherapies, chemotherapies, and/or surgery. In a preferredembodiment, the invention will provide therapeutic methods for thetreatment or amelioration of hyperproliferative disorders that have beenshown to be or may be refractory or non-responsive to other therapies.

While not wishing to be bound by any theory, it is believed that some ofthe N-oxide compounds of the invention will function as prodrugs withgreatly diminished cytotoxicity. It is believed that these N-oxidecompounds will be activated under hypoxic conditions within the targettissues (i.e., reduced at the nitrogen atom), followed by inhibition ofmicrotube formation in the mitotic spindle resulting in arrest ofdividing cells at the metaphase stage, diminishing cells' ability toreplicate. Other N-oxide compounds of the invention may have intrinsiccytotoxic activity. Since a number of pathological tissues havesignificant hypoxic components which promote hyperproliferation, it isbelieved that this portion of tissue will be preferentially targeted.

Because lower doses of chemotheraputic agents generally pose lower risksof side effects, it would be advantageous if lower doses of the agentscan be administered without compromising its efficacy. It is believedthat the N-oxide compounds, when used in combination with certainchemotherapeutic agents, will prevent or ameliorate side effects inducedby or associated with the chemotheraputic agent by decreasing theeffective dose of the agents than would otherwise be possible withoutthe use of the N-oxide compounds.

BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES

FIG. 1 shows x-ray single crystal structure of vinblastine N-oxide. Thechemical structure of the compound is also shown for comparisonpurposes.

FIG. 2A-2D show hypoxia-activated cytotoxicity of vinblastine N-oxideagainst multiple human solid tumor cell lines in vitro.

FIGS. 3A-3D show hypoxia-activated cytotoxicity of vincristine N-oxideagainst multiple human solid tumor cell lines in vitro.

FIG. 4 shows differential cytotoxic activity of vincristine N-oxideagainst viable H460 lung adenocarcinoma tumor colonies under hypoxic vs.normoxic conditions in vitro.

FIG. 5 shows activation of cytotoxicity of Vinblastine N-oxide againstH460 lung carcinoma cells is oxygen dependent. The HCR was calculated asthe IC₅₀ under normoxic conditions/IC₅₀ hypoxic conditions

FIGS. 6A-6D show chromatograms from LC/MS-MS analysis of theextracellular medium from 200 nM vinblastine N-oxide treated H460 lungadenocarcinoma tumor cells demonstrating that vinblastine N-oxide isconverted to vinblastine (parent drug) following hypoxia exposure.

FIGS. 7A and 7B show LC/MS-MS analysis of vinblastine N-oxide (filledbars) or bioreduced vinblastine (unfilled bars) from the lysates (7A) orextracellular medium (7B) of H460 cells treated with 200 nM vinblastineN-oxide exposed to normoxic or hypoxic conditions.

FIGS. 8A and 8B show LC/MS-MS analysis of vincristine N-oxide (filledbars) or bioreduced vincristine (unfilled bars) from the lysates (8A) orextracellular medium (8B) of H460 cells treated with 7 mM vincristineN-oxide exposed to normoxic or hypoxic conditions.

FIG. 9 shows the effects of vinblastine N-oxide analog (VBL-NO) orvinblastine treatment on mean percentage change in body weight loss inimmunodeficient mice (n=5 group). All agents were i.p. administered on aq3d×5 schedule at the indicated dosages.

FIG. 10 shows the effects of vincristine N-oxide analog (VCR-NO) orvincristine sulfate treatment on mean percentage change in body weightloss in tumor-bearing immunodeficient mice (n=5 group). All agents werei.v. administered on a q3d×5 schedule at the indicated dosages.

FIG. 11 shows efficacy of vincristine N-oxide analog (VCR-NO) orvincristine (VCR) in the L1210 murine leukemia model (n=8) as determinedby Tumor Growth Delay. All agents were i.v. administered on a q3d×5schedule (d. 1, 4, 7, 10, 13) at the indicated dosages.

Tumor Growth Delay:

VCR-NO 60 mg/kg=20% (P<0.05);

VCR-NO 75 mg/kg=49% (P<0.01);

Vincristine 1 mg/kg=8% (NS

FIG. 12 shows efficacy of vincristine N-oxide analog (VCR-NO) orvincristine (VCR) in the L1210 murine leukemia model (n=8) as determinedby Kaplan Meier Survival analysis. All agents were i.v. administered ona q3d×5 schedule (d. 1, 4, 7, 10, 13) at the indicated dosages.

Median Survival (Days):

Vehicle=18.1;

VCR-NO 60 mg/kg=21.7;

VCR-NO 75 mg/kg=28.2;

Vincristine 1 mg/kg=19.6

FIG. 13 shows efficacy of vinblastine N-oxide analog (VBL-NO) orvinblastine (VBL) in the L1210 murine leukemia model (n=8) as determinedby Tumor Growth Delay. All agents were i.v. administered on a q3d×5schedule (d. 1, 4, 7, 10, 13) at the indicated dosages.

Tumor Growth Delay:

VBL-NO 5 mg/kg=18% (NS);

VBL-NO 15 mg/kg=0% (NS);

VBL-NO 30 mg/kg=41% (P<0.01);

Vinblastine 5 mg/kg=34% (P<0.05)

FIG. 14 shows efficacy of vinblastine N-oxide analog (VBL-NO) orvinblastine (VBL) in the L1210 murine leukemia model (n=8) as determinedby Kaplan Meier Survival analysis. All agents were i.v. administered ona q3d×5 schedule (d. 1, 4, 7, 10, 13) at the indicated dosages.

Median Survival (Days):

Vehicle=18.1;

VBL-NO 5 mg/kg=21.3;

VBL-NO 15 mg/kg=17.8;

VBL-NO 30 mg/kg=25.5;

Vinblastine 5 mg/kg=24.3

FIG. 15 shows efficacy of vincristine N-oxide analog (VCR-NO) orvincristine (VCR) in the P388 murine leukemia model (n=8) as determinedby Tumor Growth Delay. All agents were i.v. administered on a q3d×5schedule (d. 1, 4, 7, 10, 13) at the indicated dosages.

Tumor Growth Delay:

VCR-NO 60 mg/kg=76% (P<0.001);

VCR-NO 75 mg/kg=54% (P<0.001);

Vincristine 1 mg/kg=54% (P<0.001)

FIG. 16 shows efficacy of vincristine N-oxide analog (VCR-NO) orvincristine (VCR) in the P388 murine leukemia model (n=8) as determinedby Kaplan Meier Survival analysis. All agents were i.v. administered ona q3d×5 schedule (d. 1, 4, 7, 10, 13) at the indicated dosages.

Median Survival (Days):

Vehicle=13.5;

VCR-NO 60 mg/kg=23.8;

VCR-NO 75 mg/kg=20.9;

Vincristine 1 mg/kg=20.8

FIG. 17 shows efficacy of vinblastine N-oxide analog (VBL-NO) orvinblastine (VBL) in the P388 murine leukemia model (n=8) as determinedby Tumor Growth Delay. All agents were i.v. administered on a q3d×5schedule (d. 1, 4, 7, 10, 13) at the indicated dosages.

Tumor Growth Delay:

VBL-NO 5 mg/kg=0% (NS);

VBL-NO 15 mg/kg=24% (P<0.05);

VBL-NO 30 mg/kg=92% (P<0.001);

Vinblastine 5 mg/kg=149% (P<0.001)

FIG. 18 shows efficacy of vinblastine N-oxide analog (VBL-NO) orvinblastine (VBL) in the P388 murine leukemia model (n=8) as determinedby Kaplan Meier Survival analysis. All agents were i.v. administered ona q3d×5 schedule (d. 1, 4, 7, 10, 13) at the indicated dosages.

Median Survival (Days):

Vehicle=13.5;

VBL-NO 5 mg/kg=13.4;

VBL-NO 15 mg/kg=17.8;

VBL-NO 30 mg/kg=25.9;

Vinblastine 5 mg/kg=30.8

FIG. 19 shows efficacy of vincristine N-oxide analog (VCR-NO) orvincristine (VCR) in the K562 myelogenous leukemia xenograft model innude mice (n=10) as determined by Tumor Growth Inhibition. All agentswere i.v. administered on a q3d×5 schedule (d. 1, 4, 7, 10, 13) at theindicated dosages.

Tumor Growth Inhibition:

VCR-NO 30 mg/kg=69% (P<0.01);

VCR-NO 40 mg/kg=84% (P<0.01);

Vincristine 1.5 mg/kg=95% (P<0.01)

FIG. 20 shows efficacy of vincristine N-oxide analog (VCR-NO) orvincristine (VCR) in the K562 myelogenous leukemia xenograft model innude mice (n=10) as determined by Kaplan Meier Survival analysis. Allagents were i.v. administered on a q3d×5 schedule (d. 1, 4, 7, 10, 13)at the indicated dosages.

Median Survival (Days):

Vehicle=30;

VCR-NO 30 mg/kg=48;

VCR-NO 40 mg/kg=60;

Vincristine 1.5 mg/kg=60

FIG. 21 shows efficacy of vinblastine N-oxide analog (VBL-NO) orvinblastine (VBL) in the K562 myelogenous leukemia xenograft model innude mice (n=10) as determined by Tumor Growth Inhibition. All agentswere i.v. administered on a q3d×5 schedule (d. 1, 4, 7, 10, 13) at theindicated dosages.

Tumor Growth Inhibition:

VBL-NO 25 mg/kg=77% (P<0.01);

VBL-NO 35 mg/kg=89% (P<0.01);

Vinblastine 2.5 mg/kg=77% (P<0.01)

FIG. 22 shows efficacy of vinblastine N-oxide analog (VBL-NO) orvinblastine (VBL) in the K562 myelogenous leukemia xenograft model innude mice (n=10) as determined by Kaplan Meier Survival analysis. Allagents were i.v. administered on a q3d×5 schedule (d. 1, 4, 7, 10, 13)at the indicated dosages.

Median Survival (Days):

Vehicle=30;

VBL-NO 25 mg/kg=51;

VBL-NO 35 mg/kg=57;

Vinblastine 2.5 mg/kg=60

FIG. 23 shows efficacy of vincristine N-oxide analog (VCR-NO) orvincristine (VCR) in the HL60 promyelocytic leukemia xenograft model innude mice (n=10) as determined by Tumor Growth Inhibition. All agentswere i.v. administered on a q3d×5 schedule (d. 1, 4, 7, 10, 13) at theindicated dosages.

Tumor Growth Inhibition:

VCR-NO 30 mg/kg=94% (P<0.01);

VCR-NO 40 mg/kg=96% (P<0.01);

Vincristine 1.5 mg/kg=95% (P<0.01

FIG. 24 shows efficacy of vincristine N-oxide analog (VCR-NO) orvincristine (VCR) in the HL60 promyelocytic leukemia xenograft model innude mice (n=10) as determined by Kaplan Meier Survival analysis. Allagents were i.v. administered on a q3d×5 schedule (d. 1, 4, 7, 10, 13)at the indicated dosages.

Median Survival (Days):

Vehicle=21.5;

VCR-NO 30 mg/kg=60;

VCR-NO 40 mg/kg=58;

Vincristine 1.5 mg/kg=60

FIG. 25 shows efficacy of vinblastine N-oxide analog (VBL-NO) orvinblastine (VBL) in the HL60 promyelocytic leukemia xenograft model innude mice (n=10) as determined by Kaplan Meier Survival analysis. Allagents were i.v. administered on a q3d×5 schedule (d. 1, 4, 7, 10, 13)at the indicated dosages.

Tumor Growth Inhibition:

VBL-NO 25 mg/kg=91% (P<0.01);

VBL-NO 35 mg/kg=94% (P<0.01);

Vinblastine 2.5 mg/kg=95% (P<0.01)

FIG. 26 shows efficacy of vinblastine N-oxide analog (VBL-NO) orvinblastine (VBL) in the HL60 promyelocytic leukemia xenograft model innude mice (n=10) as determined by Kaplan Meier Survival analysis. Allagents were i.v. administered on a q3d×5 schedule (d. 1, 4, 7, 10, 13)at the indicated dosages.

Median Survival (Days):

Vehicle=21.5;

VBL-NO 25 mg/kg=60;

VBL-NO 35 mg/kg=60;

Vinblastine 2.5 mg/kg=60

FIG. 27 shows efficacy of vincristine N-oxide analog (VCR-NO) as singleagent or in combination with CPT-11 in the HT29 colon xenograft model innude mice model (n=10) as determined by Tumor Growth Delay. VCR-NO wasi.v. administered on a q3d×5 schedule (d. 1, 4, 7, 10, 13) at 15 mg/kg.CPT-11 was i.p. administered at 100 mg/kg on a q week×3 schedule.

Tumor Growth Delay:

VCR-NO 15 mg/kg=52%;

CPT-11 100 mg/kg=52%;

VCR-NO 25 mg/kg+CPT-11 100 mg/kg=111%

FIG. 28 shows efficacy of vincristine N-oxide analog (VCR-NO) as singleagent or in combination with CPT-11 in the HT29 colon xenograft model innude mice model (n=10) as determined by Kaplan Meier Survival analysis.VCR-NO was i.v. administered on a q3d×5 schedule (d. 1, 4, 7, 10, 13).CPT-11 was i.p. administered on a q week×3 schedule at the indicateddosages.

Median Survival (Days):

Vehicle=26.0

VCR-NO 15 mg/kg=39.6

VCR-NO 25 mg/kg=37.1

CPT-11 50 mg/kg=44.7

CPT-11 100 mg/kg=39.6

VCR-NO 15 mg/kg+CPT-11 50 mg/kg=41.5

VCR-NO 25 mg/kg+CPT-11 50 mg/kg=45.4

VCR-NO 15 mg/kg+CPT-11 100 mg/kg=55.0

VCR-NO 25 mg/kg+CPT-11 100 mg/kg=47.4

FIG. 29 shows efficacy of vinblastine N-oxide analog (VBL-NO) as singleagent or in combination with CPT-11 in the HT29 colon xenograft model innude mice model (n=10) as determined by Tumor Growth Delay. VBL-NO wasi.v. administered on a q3d×5 schedule (d. 1, 4, 7, 10, 13) at 20 mg/kg.CPT-11 was i.p. administered at 50 mg/kg on a q week×3 schedule.

Tumor Growth Delay:

VBL-NO 20 mg/kg=46%

CPT-11 50 mg/kg=34%

VBL-NO 20 mg/kg+CPT-11 50 mg/kg=83%

FIG. 30 shows efficacy of vinblastine N-oxide analog (VBL-NO) as singleagent or in combination with CPT-11 in the HT29 colon xenograft model innude mice model (n=10) as determined by Kaplan Meier Survival analysis.VBL-NO was i.v. administered on a q3d×5 schedule (d. 1, 4, 7, 10, 13).CPT-11 was i.p. administered on a q week×3 schedule at the indicateddosages.

Median Survival (Days):

Vehicle=24.7

VBL-NO 10 mg/kg=22.5

VBL-NO 20 mg/kg=36.1

CPT-11 50 mg/kg=33.1

CPT-11 100 mg/kg=39.5

VBL-NO 10 mg/kg+CPT-11 50 mg/kg=38.9

VBL-NO 20 mg/kg+CPT-11 50 mg/kg=45.1

VBL-NO 10 mg/kg+CPT-11 100 mg/kg=38.0

VBL-NO 20 mg/kg+CPT-11 100 mg/kg=44.8

DETAILED DESCRIPTION OF THE INVENTION

One aspect of the invention is drawn to vinca alkaloid N-oxides havingFormula I:

wherein R₁-R₈, R′₁-R′₈, R″₃, R″₅, and n are as defined above, or apharmaceutically acceptable salt thereof.

According to another aspect of the invention, a therapeuticallyeffective amount of a compound having Formula I, or a pharmaceuticallyacceptable salt thereof, and at least one other active agent is providedin the form of a pharmaceutical composition having at least onepharmaceutically acceptable carrier. In certain instances, the at leastone other active agent is a chemotherapeutic agent (including an activevitamin D compound). Compounds having Formula I may be formulated in asingle formulation with the other active agent(s), or formulatedindependently.

According to one aspect of the invention, methods for treating,ameliorating, or preventing hyperproliferative disorders are provided,wherein a therapeutically effective amount of a compound having FormulaI, or a pharmaceutically acceptable salt thereof, is administered to ananimal in need thereof. In certain aspects of the invention, thehyperproliferative disorder is cancer.

A further aspect of the invention relates to methods for treating,ameliorating, or preventing a hyperproliferative disorder comprisingadministering a therapeutically effective amount of a compound havingFormula I, or a pharmaceutically acceptable salt thereof, in combinationwith at least one other active agent or treatment to a patient in needthereof. In certain embodiments, combinations of a compound havingFormula I with a chemotherapeutic agent are administered. In oneembodiment, the chemotherapeutic agent is selected from gemcitabine andirinotecan.

Hyperproliferative disorders which can be treated with the compoundshaving Formula I include any hypoxia-aggravated hyperproliferativedisease and/or disorder, such as any number of cancers. Generally, suchcancers include, without limitation, cancers of the bladder, brain,breast, cervix, colon, endometrium, esophagus, head and neck, kidney,larynx, liver, lung, oral cavity, ovaries, pancreas, prostate, skin,stomach, and testis. Certain of these cancers may be more specificallyreferred to as acute and chronic lymphocytic leukemia, acutegranulocytic leukemia, adrenal cortex carcinoma, bladder carcinoma,breast carcinoma, cervical carcinoma, cervical hyperplasia,choriocarcinoma, chronic granulocytic leukemia, chronic lymphocyticleukemia, colon carcinoma, endometrial carcinoma, esophageal carcinoma,essential thrombocytosis, genitourinary carcinoma, hairy cell leukemia,head and neck carcinoma, Hodgkin's disease, Kaposi's sarcoma, lungcarcinoma, lymphoma, malignant carcinoid carcinoma, malignanthypercalcemia, malignant melanoma, malignant pancreatic insulinoma,medullary thyroid carcinoma, melanoma, multiple myeloma, mycosisfungoides, myeloid and lymphocytic leukemia, neuroblastoma,non-Hodgkin's lymphoma, osteogenic sarcoma, ovarian carcinoma,pancreatic carcinoma, polycythemia vera, primary brain carcinoma,primary macroglobulinemia, prostatic carcinoma, renal cell carcinoma,rhabdomyosarcoma, skin cancer, small-cell lung carcinoma, soft-tissuesarcoma, squamous cell carcinoma, stomach carcinoma, testicularcarcinoma, thyroid carcinoma, and Wilms' tumor. In one embodiment, thecancer is a solid tumor. In another embodiment, the cancer is selectedfrom the group consisting of colon cancer, brain cancer, glioma,multiple myeloma, head and neck cancer hepatocellular cancer, melanoma,ovarian cancer, cervical cancer, renal cancer, and non-small cell lungcancer.

Animals which may be treated according to the present invention includeall animals which may benefit from administration of compounds havingFormula I. Such animals include humans, pets such as dogs and cats, andveterinary animals such as cows, pigs, sheep, goats and the like.

The term “alkyl” as used herein refers to an unsaturated acyclichydrocarbon radical. The term “lower alkyl” refers to acyclichydrocarbon radicals containing from about 2 to about 10 carbon atoms,preferably from about 2 to about 8 carbon atoms and more preferably 1 toabout 6 carbon atoms. Examples of suitable alkyl radicals includemethyl, ethyl, propyl, butyl, isobutyl, pentyl, 2-methylbutyl,3-methylbutyl, hexyl, heptyl, and octyl, and the like.

The term “alkoxy” means a straight, branched or cyclic hydrocarbonconfiguration and combinations thereof, including from 1 to 20 carbonatoms, preferably from 1 to 8 carbon atoms, more preferably from 1 toabout 4 carbon atoms, and an oxygen atom at the point of attachment.Suitable alkoxy groups include methoxy, ethoxy, n-propoxy, isopropoxy,n-butoxy, iso-butoxy, sec-butoxy, tert-butoxy, cyclopropyloxy,cyclohexyloxy, and the like. “Lower alkoxy” refers to alkoxy groupshaving from 1 to 4 carbon atoms.

The term “acyl” or “alkanoyl means an alkyl group attached to a carbonylgroup.

The term “halogenoacyl” means an acyl group substituted with one or morehalogen groups (e.g. F, Cl, Br and I groups), including trifluoroacetyl,pentafluoropropionyl and the like.

The term “non-N-oxide” as used herein refers to an amine compound thatis not oxidized at the nitrogen atom. As an example, vinblastine is thenon-N-oxide form of vinblastine N-oxide.

The term “pharmaceutical composition” as used herein, is to beunderstood as defining compositions of which the individual componentsor ingredients are themselves pharmaceutically acceptable, e.g., whereoral administration is foreseen, acceptable for oral use; where topicaladministration is foreseen, topically acceptable; and where intravenousadministration is foreseen, intravenously acceptable.

As used herein, the term “therapeutically effective amount” refers tothat amount of the therapeutic agent sufficient to result inamelioration of one or more symptoms of a disorder, or preventadvancement of a disorder, or cause regression of the disorder. Forexample, with respect to the treatment of cancer, a therapeuticallyeffective amount preferably refers to the amount of a therapeutic agentthat decreases the rate of tumor growth, decreases tumor mass, decreasesthe number of metastases, increases time to tumor progression, orincreases survival time by at least 5%, preferably at least 10%, atleast 15%, at least 20%, at least 25%, at least 30%, at least 35%, atleast 40%, at least 45%, at least 50%, at least 55%, at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, or at least 100%.

The terms “prevent,” “preventing,” and “prevention,” as used herein,refer to a decrease in the occurrence of pathological cells (e.g.,hyperproliferative or neoplastic cells) in an animal. The prevention maybe complete, e.g., the total absence of pathological cells in a subject.The prevention may also be partial, such that the occurrence ofpathological cells in a subject is less than that which would haveoccurred without the present invention.

Compounds having Formula I can be provided as pharmaceuticallyacceptable salts. Examples of pharmaceutically acceptable salts (i.e.,addition salts) include inorganic and organic acid addition salts suchas hydrochloride, hydrobromide, phosphate, sulphate, citrate, lactate,tartrate, maleate, fumarate, mandelate, benzoate and oxalate; andinorganic and organic base addition salts with bases such as sodiumhydroxy, Tris(hydroxymethyl)aminomethane (TRIS, tromethane) andN-methyl-glucamine. Although the salts typically have similarphysiological properties compared to the free base, certain acidaddition salts may demonstrate preferred physicochemical properties,e.g., enhanced solubility, improved stability. Certain salts exhibitless hygroscopicity. Some salts may be more easily crystallized thanothers. Other salts form free flowing powders. For example, in oneembodiment, vinblastine N-oxide dihydrochloride forms a stable clearsolution at about 20 mg/mL in de-ionized water and in 5% dextrose/watersolutions. One particular pharmaceutically acceptable salt is derivedfrom maleic acid, the salt being either a hydrogen maleate or adimaleate salt. Another particular pharmaceutically acceptable salt ishydrochloride salts. In one embodiment, the salt is vinblastine N-oxidedihydrochloride. In another embodiment, the salt is vincristine N-oxidedihydrochloride.

Certain of the compounds of the present invention may exist asstereoisomers including optical isomers as well as the individualenantiomers that may be separated according to methods that are wellknown to those of ordinary skill in the art. Optical purity orenantiomeric excess (ee) may range from 0%-100%. The invention includesall stereoisomers and both the racemic mixtures of such stereoisomers aswell as the individual enantiomers that may be separated according tomethods that are well known to those of ordinary skill in the art.Certain of the compounds of the present invention may also exist asdiasteroisomers wherein one or more substituents on the vinca alkaloidanalog contain one or more chiral centers.

In certain embodiments, the N-oxide formation creates a new chiralcenter with the formation of individual enantiomers (e.g., in astereoselective N-oxidation of an achiral vinca alkaloid or analog),mixture of enantiomers (e.g., in a non-stereoselective oxidation of anachiral vinca alkaloid or analog), individual diasteroisomers (e.g., ina stereoselective N-oxidation of an enantiomerically pure vinca alkaloidor analog), or mixtures of diasteroisomers (e.g., in anon-stereoselective N-oxidation of an enantiomeric mixture of a vincaalkaloid or analog). Thus, the invention includes all N-oxide mixturesof enantiomers and diasteroisomers as well as individual diasteroisomersand enantiomers that may be prepared using stereoselective reactions orseparated according to methods that are well known to those of ordinaryskill in the art.

In certain embodiments of the invention, compounds having Formula I areadministered in combination with one or more other active agents (e.g.,chemotherapeutic agents) or treatments. By way of non-limiting example,a patient may be treated for a hyperproliferative disorder, such ascancer, by the administration of a therapeutically effective amount of acompound having Formula I in combination with radiotherapyagent/treatment or the administration of a chemotherapeutic agent.

In other embodiments, compounds of the invention are administered incombination with agents, such as anti-angiogenic agents, that blockinhibit or modulate tumor neovascularization. In preferred embodiments,anti-angiogenesis agents can be any anti-angiogenesis agent which isused, has been used, or is known to be useful for the treatment ofhyperproliferative disorders. Examples of anti-angiogenesis agentsinclude bevacizumab (Avastin™), VEGF-TRAP, anti-VEGF-receptorantibodies, angiostatin, endostatin, batimastat, captopril, cartilagederived inhibitor, genistein, interleukin 12, lavendustin,medroxypregesterone acetate, recombinant human platelet factor 4,tecogalan, thrombospondin, TNP-470, VEGF antagonists, anti-VEGFmonoclonal antibody, soluble VEGF-receptor chimaeric protein, antisenseoligonucleotides, antisense oligodexoynucleotides, siRNAs, anti-VEGFaptamers, pigment epithelium derived factor, a tyrosine kinaseinhibitor, an inhibitor of epidermal-derived growth factor, an inhibitorof fibroblast-derived growth factor, an inhibitor of platelet derivedgrowth factor, an MMP (matrix metalloprotease) inhibitor, an integrinblocker, interferon-α, pentosan polysulfate, a cyclooxygenase inhibitor,carboxyamidotriazole, combretastatin A-4, squalamine,6-O-chloroacetyl-carbonyl)-fumagillol, thalidomide, troponin-1,indolinethiones, pyridopyrimidines, quinoazolines,phenyl-pyrrolo-pyrimidines, trastuzumab, calcium influx inhibitor (CAI),neomycin, squalamine, marimastat, prinomastat (AG-3340), metastat(COL-3) and cinnoline derivatives. Additional anti-angiogenic compoundsthat may be administered in combination with the compounds of thepresent invention are described in U.S. Pat. Nos. 5,192,744, 5,426,100,5,733,876, 5,840,692, 5,854,205, 5,990,280, 5,994,292, 6,342,219,6,342,221, 6,346,510, 6,479,512, 6,719,540, 6,797,488, 6,849,599,6,869,952, 6,887,874, 6,958,340 and 6,979,682.

In certain embodiments, the compounds of the present invention areadministered in combination with a vascular targeting agent (also knownas vascular damaging agents). In one embodiment, the vascular targetingagent is for the treatment of malignant or non-malignant vascularproliferative disorders. In other embodiments, vascular targeting agentscan be any vascular targeting agent which is used, has been used, or isknown to be useful for the treatment of hyperproliferative disorders.Examples of vascular targeting agents that may be administered incombination with the compounds of the present invention include DMXAA5,6-dimethylxanthenone-4-acetic acid, ZD6126,(5S)-5-(acetylamino)-9,10,11-trimethoxy-6,7-dihydro-5H-dibenzo[a,c]cyclohepten-3-yldihydrogen phosphate, also known as N-acetylcolchinol-O-phosphate (see,for example, U.S. Pat. No. 6,906,048); functionalized stilbenederivatives such as combretastatin A4 and its prodrugs (see, e.g., U.S.Pat. Nos. 6,919,324 and 6,773,702); dioleoyltrimethyl-ammonium propane(DOTAP), N-[1-(2,3-dioleoyloxy)-propyl]-N,N,N-trimethylammonium chloride(DOTMA), dimethyldioctadecylammonium bromide (DDAB),1,2-dimyristyloxypropyl-3-dimethylhydroxyethyl (DMRIE),dioleoyl-3-dimethylammonium propane (DODAP),N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), orN-(1-(2,3-dioleyloxy)propyl)-N-(2-(sperminecarboxamido)ethyl)-N,N-dimethylammonium trifluoroacetate (DOSPA), or any other natural or syntheticcationic lipids, including, for example, dioleoylphosphatidyl-choline(DOPC), dipalmitoylphosphatidylcholine (DPPC),disteroylphosphatidylcholine (DSPC), dimyristoylphosphatidylcholine(DMPC), or 1,2-sn-dioleoylphosphatidylcholine (DOPE), or any othernatural or synthetic electrostatically neutral lipids (see, for example,U.S. Pat. No. 6,680,068); vascular targeting agents which incorporatebenzo[b]thiophene, indole, and benzofuran molecular skeletons such asthose described in U.S. Pat. No. 6,593,374.

In other embodiments, the compounds of the present invention areadministered in combination with a hypoxia-inducible factor 1 (HIF1)inhibitor. In one embodiment, the HIF1 inhibitor is for the treatment ofmalignant or non-malignant vascular proliferative disorders. In otherembodiments, HIF1 inhibitors can be any HIF1 inhibitor which is used,has been used, or is known to be useful for the treatment ofhyperproliferative disorders. Examples of HIF1 inhibitors suitable foruse in combination with compounds of the present invention includetopotecan, P13 kinase inhibitors; LY294002; rapamycin; histonedeacetylase inhibitors such as[(E)-(1S,4S,10S,21R)-7-[(Z)-ethylidene]-4,21-diisopropyl-2-oxa-12,13-dithia-5,8,20,23-tetraazabicyclo-[8,7,6]-tricos-16-ene-3,6,9,19,22-pentanone(FR901228, depsipeptide); heat shock protein 90 (Hsp90) inhibitors suchas geldanamycin, 17-allylamino-geldanamycin (17-AAG), and othergeldanamycin analogs, and radicicol and radicicol derivatives such asKF58333; genistein; indanone; staurosporin; protein kinase-1 (MEK-1)inhibitors such as PD98059 (2′-amino-3′-methoxyflavone); PX-12(1-methylpropyl 2-imidazolyl disulfide); pleurotin PX478; quinoxaline1,4-dioxides; sodium butyrate (NaB); sodium nitropurruside (SNP) andother NO donors; microtubule inhibitors such as novobiocin, panzem(2-methoxyestradiol or 2-ME2), vincristines, taxanes, epothilones,discodermolide, and derivatives of any of the foregoing; coumarins;barbituric and thiobarbituric acid analogs; camptothecins; and YC-1. SeeU.S. Pat. No. 6,979,675.

In certain embodiments, the compounds of the present invention areadministered in combination with an Hsp90 inhibitor. In one embodiment,the Hsp90 inhibitor is for the treatment of malignant or non-malignantvascular proliferative disorders. In other embodiments, Hsp90 inhibitorscan be any Hsp90 inhibitor which is used, has been used, or is known tobe useful for the treatment of hyperproliferative disorders. Examples ofHsp90 inhibitors that may be combined with the compounds of the presentinvention include geldanamycin, 17-allylamino-17-demethoxygeldanamycin,geldanamycin derivatives such as those described in U.S. Pat. No.6,890,917, dexamethasone and benzoquinone ansamycins such as thosedescribed in U.S. Pat. No. 6,872,715. Additional Hsp90 inhibitors aredisclosed in U.S. Pat. Nos. 6,613,780, 6,281,229 and 6,903,116.

In other embodiments, the compounds of the present invention areadministered in combination with an inhibitor of tyrosine and/orserine/threonine kinases and tyrosine kinase receptors involved incellular signaling. These include tyrosine kinase inhibitors of Src,Abl, Platelet Derived Growth Factor Receptors, Vascular EndothelialGrowth Factor Receptors, c-Met, Fibroblast Growth Factor receptors,Epidermal Growth Factor Receptors, Insulin Growth Factor Receptors,mTOR, Flt-3, CSF-1 Receptor, AKT, Polo kinases, Aurora Kinases, STAT-3,PI-3 Kinase, Ras, Raf and Mitogen Activated Kinases, MEK, ERK. Examplesof tyrosine kinase and serine/threonine kinase inhibitors include (butnot limited to): AMG706, ZA6474, BAY 43-9006, Dasatinib, CEP-701, XL647,XL999, Lapatinb, MLN518/CT53518, PKC412, ST1571, AMN107, AEE 788,OSI-930, OSI-817, SU11248, AG-03736, GW-786034m, CEP-7055.

In other embodiments, the compounds of the present invention areadministered in combination with HDAC inhibitors. Examples include (butnot limited to) SAHA, MS-275, MGCD0103, LBH589, PXD101, FK228.

In other embodiments, the compounds of the present invention areadministered in combination with proteasome inhibitors such as Velcade.

In other embodiments, the compounds of the present invention areadministered in combination with pro-apoptotic agents such as TRAIL,anti-DR4/DR5 (TRA8) antibodies, IAP, Survivin or small molecules thatstimulate caspase activation.

In other embodiments, the compounds of the present invention areadministered in combination with inhibitors of cell cycle regulatorssuch as CDK inhibitors.

“In combination” refers to the use of more than one treatment. The useof the term “in combination” does not restrict the order in whichtreatments are administered to a subject being treated for ahyperproliferative disorder. A first treatment can be administered priorto, concurrently with, after, or within any cycling regimen involvingthe administration of a second treatment to a subject with ahyperproliferative disorder. For example, the first treatment can beadministered 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or12 weeks before a treatment; or the first treatment can be administered5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours,6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after asecond treatment. Such treatments include, for example, theadministration of compounds having Formula I in combination with one ormore chemotherapeutic agents or radiotherapeutic agents/treatments.

The term “chemotherapeutic agent,” as used herein, is intended to referto any chemotherapeutic agent known to those of skill in the art to beeffective for the treatment, prevention or amelioration ofhyperproliferative disorders such as cancer. Chemotherapeutic agentsinclude, but are not limited to, small molecules, synthetic drugs,peptides, polypeptides, proteins, nucleic acids (e.g., DNA and RNApolynucleotides including, but not limited to, antisense nucleotidesequences, triple helices and nucleotide sequences encoding biologicallyactive proteins, polypeptides or peptides), antibodies, synthetic ornatural inorganic molecules, mimetic agents, and synthetic or naturalorganic molecules. Any agent which is known to be useful, or which hasbeen used or is currently being used for the treatment or ameliorationof a hyperproliferative disorder can be used in combination with acompound having Formula I. See, e.g., Hardman et al., eds., 2002,Goodman & Gilman's The Pharmacological Basis Of Therapeutics 10th Ed,Mc-Graw-Hill, New York, N.Y. for information regarding therapeuticagents which have been or are currently being used for the treatment oramelioration of a hyperproliferative disorder.

Particular chemotherapeutic agents useful in the methods andcompositions of the invention include alkylating agents,antimetabolites, anti-mitotic agents, epipodophyllotoxins, antibiotics,hormones and hormone antagonists, enzymes, platinum coordinationcomplexes, anthracenediones, substituted ureas, methylhydrazinederivatives, imidazotetrazine derivatives, cytoprotective agents, DNAtopoisomerase inhibitors, biological response modifiers, retinoids,therapeutic antibodies, differentiating agents, immunomodulatory agents,angiogenesis inhibitors and anti-angiogenic agents.

Certain chemotherapeutic agents include, but are not limited to,abarelix, aldesleukin, alemtuzumab, alitretinoin, allopurinol,altretamine, amifostine, anastrozole, arsenic trioxide, asparaginase,BCG live, bevaceizumab, bexarotene, bleomycin, bortezomib, busulfan,calusterone, camptothecin, capecitabine, carboplatin, carmustine,celecoxib, cetuximab, chlorambucil, cinacalcet, cisplatin, cladribine,cyclophosphamide, cytarabine, dacarbazine, dactinomycin, darbepoetinalfa, daunorubicin, denileukin diftitox, dexrazoxane, docetaxel,doxorubicin, dromostanolone, Elliott's B solution, epirubicin, epoetinalfa, estramustine, etoposide, exemestane, filgrastim, floxuridine,fludarabine, fluorouracil, fulvestrant, gemcitabine, gemtuzumabozogamicin, gefitinib, goserelin, hydroxyurea, ibritumomab tiuxetan,idarubicin, ifosfamide, imatinib, interferon alfa-2a, interferonalfa-2b, irinotecan, letrozole, leucovorin, levamisole, lomustine,meclorethamine, megestrol, melphalan, mercaptopurine, mesna,methotrexate, methoxsalen, methylprednisolone, mitomycin C, mitotane,mitoxantrone, nandrolone, nofetumomab, oblimersen, oprelvekin,oxaliplatin, paclitaxel, pamidronate, pegademase, pegaspargase,pegfilgrastim, pemetrexed, pentostatin, pipobroman, plicamycin,polifeprosan, porfimer, procarbazine, quinacrine, rasburicase,rituximab, sargramostim, streptozocin, talc, tamoxifen, tarceva,temozolomide, teniposide, testolactone, thioguanine, thiotepa,topotecan, toremifene, tositumomab, trastuzumab, tretinoin, uracilmustard, valrubicin, vinblastine, vincristine, vinorelbine, andzoledronate. In certain embodiments, chemotherapeutic agents areselected from gemcitabine and irinotecan.

Chemotherapeutic agents may be administered at doses that are recognizedby those of skill in the art to be effective for the treatment of thehyperproliferative disorder. In certain embodiments, chemotherapeuticagents may be administered at doses lower than those used in the art dueto the additive or synergistic effect of the compounds having Formula I.

Accordingly, the present invention relates to a method for preventing orameliorating side effects induced by or associated with chemotherapy byusing lower effective doses of the chemotherapeutic agents than wouldotherwise be possible without the use of the compounds of Formula I. Inparticular, the method relates to amelioration, prevention of sideeffects induced by or associated with the chemotherapy of a variety ofcancers including, but not limited to, brain cancer, breast cancer,gastrointestinal cancers comprising colon, colorectal, esophageal,gastric, hepatocellular, pancreatic and rectal cancers, genitourinarycancers comprising bladder, prostate, renal cell and testicular cancers,gynecologic cancers comprising cervical, endometrial, ovarian anduterine cancers, head and neck cancer, leukemias comprising acutelymphoblastic, acute myelogenous, acute promyelocytic, chroniclymphocytic, chronic myelogenous, and hairy cell leukemias,non-small-cell and small-cell lung cancers, Hodgkin's and non-Hodgkin'slymphomas, melanoma, multiple myeloma and sarcoma.

Therapeutic agents useful in the methods and compositions of theinvention include active vitamin D compound or mimics thereof,antineoplastic agents (e.g., actinomycin D, vincristine, vinorelbine,topoisomerase 1 inhibitors (camptothecin and analogs such as irinotecan,SN-38, topotecan, 9-aminocamptothecin, 10-aminocamptothecin,10,11-methylenedioxycamptothecin), azacitidine (5-azacytidine, 5AzaC),thalidomide vinblastine, methotrexate, azathioprine, fluorouracil,doxorubicin, mitomycin, taxanes (docetaxel, paclitaxel)), angiogenicinhibitors (e.g., VEGF-TRAP, angiostatin, endostatin, aptamer antogonistof VEGF, batimastat, captopril, cartilage derived inhibitor, genistein,interleukin 12, lavendustin, medroxypregesterone acetate, recombinanthuman platelet factor 4, tecogalan, thrombospondin and TNP-470),serine/threonine kinase inhibitors, tyrosine kinase inhibitors, HDACinhibitors, Proteasome inhibitors, CDK inhibitors, HSP inhibitors,vasodilators (e.g., nitrates, calcium channel blockers), anticoagulants(e.g., heparin), anti-platelet agents (e.g., aspirin, blockers ofIIb/IIIa receptors, clopidogrel), anti-thrombins (e.g., hirudin,iloprost), immunosuppressants (e.g., sirolimus, tranilast,dexamethasone, tacrolimus, everolimus, A24), collagen synthetaseinhibitors (e.g., halofuginone, propyl hydroxylase, C-proteinaseinhibitor, metalloproteinase inhibitor), anti-inflammatories (e.g.,corticosteroids, non-steroidal anti-inflammatory drugs), 17β-estradiol,angiotensin converting enzyme inhibitors, colchicine, fibroblast growthfactor antagonists, histamine antagonists, lovastatin, nitroprusside,phosphodiesterase inhibitors, prostaglandin inhibitors, suramin,serotonin blockers, thioprotease inhibitors, platelet-derived growthfactor antagonists, nitric oxide, and angiopeptin. In one embodiment,the therapeutic agent is a taxane, e.g., paclitaxel or docetaxel.

Administration of some topoisomerase 1 inhibitors to patients in need ofsuch treatment is known to cause serious adverse events. For example,various serious adverse events are known to be associated with theadministration of CAMPTOSAR® to patients, including early and late stagediarrhea. See CAMPTOSAR® Product Label, Pfizer, Inc.

In three open-label studies, CAMPTOSAR® was administered in repeated6-week cycles consisting of a 90-minute intravenous infusion once weeklyfor 4 weeks, followed by a 2-week rest period. Starting doses ofCAMPTOSAR® for these trials were 100, 125, or 150 mg/m² but the 150mg/m² dose was poorly tolerated due to unacceptably high rates of grade4 late diarrhea and febrile neutropenia.

In a multicenter study that enrolled 166 patients from 30 institutionsevaluating CAMPTOSAR® as second-line treatment for recurrent orprogressive metastatic colorectal cancer (referred to as Study 3 in theCAMPTOSAR® product label), the initial dose was 125 mg/m² but wasreduced to 100 mg/m² because the toxicity seen in the 125 mg/m² dose wasperceived to be greater than those seen in previous studies.

Data from an open-label, single-agent, single arm, multicenter, clinicalstudy involving a total of 132 patients is cited as supporting a onceevery-3-week dosage schedule of irinotecan in the treatment of patientswith metastatic cancer of the colon or rectum that recurred orprogressed following treatment with 5-FU. Patients received a startingdose of 350 mg/m² given by 30-minute intravenous infusion once every 3weeks. Among the 132 previously treated patients in this trial, theintent-to-treat response rate was 12.1% (95% CI, 7.0% to 18.1%).

According to the product label, injection of CAMPTOSAR can induce bothearly and late forms of diarrhea that appear to be mediated by differentmechanisms. Early diarrhea (occurring during or shortly after infusionof CAMPTOSAR) is cholinergic in nature. It is usually transient and onlyinfrequently is severe. It may be accompanied by symptoms of rhinitis,increased salivation, miosis, lacrimation, diaphoresis, flushing, andintestinal hyperperistalsis that can cause abdominal cramping. Latediarrhea (generally occurring more than 24 hours after administration ofCAMPTOSAR) can be prolonged, may lead to dehydration and electrolyteimbalance, and can be life threatening. Patients with severe diarrheashould be carefully monitored and given fluid and electrolytereplacement if they become dehydrated. National Cancer Institute (NCI)grade 3 diarrhea is defined as an increase of 7 to 9 stools daily, orincontinence, or severe cramping and NCI grade 4 diarrhea is defined asan increase of bloody stool, or need for parenteral support. If grade 3or 4 late diarrhea occurs, administration of CAMPTOSAR should be delayeduntil the patient recovers and subsequent doses should be decreased.Myelosuppression. Deaths due to sepsis following severe myelosuppressionhave been reported in patients treated with CAMPTOSAR. The CAMPTOSARproduct label recommends temporarily omission of the therapy ifneutropenic fever occurs or if the absolute neutrophil count drops below1,000/mm³. The label further instructs that even after the patientrecovers to an absolute neutrophil count >1,500/mm³, subsequent doses ofCAMPTOSAR should be reduced depending upon the level of myelosuppressionobserved.

Since lower doses of drugs generally pose lower risks of these sideeffects, it would be advantageous if lower doses of irinotecan can beadministered without compromising its efficacy. One possible solution isto administer irinotecan in combination with a vinca alkaloid N-oxide.In certain embodiments of the present invention, a vinca alkaloidN-oxide (e.g., vincristine N-oxide) is administered to a subject incombination with topoisomerase 1 inhibitor (e.g., a camptothecin analogsuch as irinotecan) such that a synergistic anti-hyperproliferativeeffect is produced. A “synergistic anti-hyperproliferative effect”refers to a greater-than-additive anti-hyperproliferative effect whichis produced by a combination of two drugs, and which exceeds that whichwould otherwise result from individual administration of either drugalone. Administration of certain doses of a vinca alkaloid N-oxide incombination with certain doses of a topoisomerase 1 inhibitor (e.g.,irinotecan) unexpectedly resulted in an enhanced anti-hyperproliferativeeffect by providing greater efficacy than would otherwise result fromuse of either of the two agents alone. For example, in vivoadministration of certain doses of vincristine N-oxide or vinblastineN-oxide with certain doses irinotecan produced enhancedanti-hyperproliferative effect beyond that which would be expected fromthe individual components (as measured by, for example, increased tumorgrowth delay). Moreover, in vivo administration of certain doses ofirinotecan in combination with a vinca alkaloid N-oxide producedantihypreproliferative effect greater than that produced by twice thedose of irinotecan alone. Therefore, lower doses of one or both of thetwo agents may be used in treating hyperproliferative disorders,resulting in increased therapeutic efficacy, and/or decreasedside-effects such as grade 4 late diarrhea and febrile neutropenia.

Other chemotherapeutic agents are also known to cause some unwanted sideeffects. Some of these side effects may be mild and treatable (such asdizziness, nausea, and some vomiting and/or diarrhea) while others aresevere, life-threatening or even lethal. Among the more serious sideeffects are pulmonary toxicities that may lead to grades III-IVpneumonia, acute respiratory distress syndrome, or pulmonary fibrosis.Several cytotoxic drugs, including taxanes, bleomycin, methotrexate,busulfan, and the nitrosoureas may cause interstitial pneumonitis,alveolitis and pulmonary fibrosis. Administration of multiple cytotoxicdrugs and pre-existing lung disease may potentiate pulmonary toxicity.Gucalap, R. and Dutcher, J. “Oncologic emergencies,” in Harrison'sPrinciples of internal medicine, Vol. 1, Fauci, A. S. et al., eds.,14^(th) ed., McGraw-Hill, New York, N.Y., pp. 627-634 (1998).

Acute or subacute pneumonia generally affects the cells that line thealveoli, which are small sacs in the lungs that are responsible forexchanging oxygen from the air with carbon dioxide in the blood.Inflammation of these sensitive structures makes gas (oxygen and carbondioxide) exchange less efficient, reducing the amount of oxygen that isabsorbed from the air and delivered to the body. Various drugs used forthe chemotherapy of cancer can damage lung tissues resulting inpneumonia. For example, 15% of patients suffering from head and neckcancer and treated with paclitaxel, a taxane similar to docetaxel (175mg/m² over 3 hours on day 1), ifosfamide (1000 mg/m² over 2 hours ondays 1-3), cisplatin (60 mg/m² IV day 1, repeated every 3-4 weeks), andmesna (600 mg/m² on days 1-3 in two divided doses, 400 mg/m² IV beforeifosfamide and 200 mg/m² IV 4 hours after ifosfamide) requiredhospitalization due to pneumonia. Shin, D. M. et al., J. Clin. Oncol.16: 1325-30 (1998). Also, 7% of acute myelogenous patients treated withgemtuzumab (9 mg/m² IV over 2 hours, two doses with 14 days between thedoses) suffered from grade III or IV pneumonia. Product package insertfor Mylotarg™, Wyeth-Ayerst Pharmaceuticals, Inc. Moreover, 7% ofpatients with myeloid blast crisis treated with once a day oral dose ofeither 400 mg or 600 mg imatinib mesylate (Gleevec®) developed grade IIIor IV pneumonia. Product package insert for Gleevac®, NovartisPharmaceutical Corporation. In two single-arm open-label studies offludarabine phosphate (Fludara®) in patients with refractory chroniclymphocytic leukemia, 16% of patients receiving 22-40 mg/m² dailyFludara® injections for five days every 28 days and 22% of patientsreceiving 15-25 mg/m² daily Fludara® injection for five days every 28days developed pneumonia. Product insert for Fludara®, BerlexLaboratories, Richmond, Calif. Also, one of 44 cervical cancer patientstreated with paclitaxel (135 mg/m² IV over 24 hours day 1), followed bycisplatin (75 mg/m² IV day 2, repeat every 21 days) developed grade IIIor IV pneumonia. Rose, P. G. et al., J. Clin. Oncol., 17: 2678-80(1999). Other cancer drugs that have been implicated to cause pneumoniawith grade III or IV toxicity include alemtuzmab (Campath®). Indeed, theproduct package insert of Campath® indicates that prophylaxis directedagainst Pneumocystis carinii pneumonia used in connection with Campath®treatment decreases, but does not eliminate, the occurrence of thisinfection.

Another example of pulmonary toxicity induced by or associated withchemotherapy is pulmonary fibrosis. Pulmonary fibrosis is thedevelopment of fibrous scar tissue in the lungs. Lung tissue is normallyvery elastic and expands as one breathes in order to provide a largerspace for air. As scar tissue builds up in the lung, in some cases as aresult of acute inflammation, the air sacs of the lungs become graduallyreplaced by fibrotic tissue. When the scar forms, the tissue becomesthicker causing an irreversible loss of the tissue's ability to transferoxygen into the bloodstream. Various drugs used for the chemotherapy ofcancer cause pulmonary fibrosis. Bleomycin (BLM) is known to inducepulmonary complications. Indeed, 7 of 148 testicular cancer patientstreated with bleomycin (30 units IV, weekly), etoposide (100 mg/m²/d IVdays 1-5) and cisplatin (20 mg/m²/d IV days 1-5), repeat cycle every 3weeks for four 3-week periods, experienced grade III-IV respiratorytoxicity with 3 patient deaths due to pulmonary toxicity. Nichols, J.R., et al. J. Clin. Oncol. 16: 1287-93 (1998).

Acute Respiratory Distress Syndrome (“ARDS”) is a life-threateningcondition in which inflammation of the lungs and accumulation of fluidin the air sacs (alveoli) leads to low blood oxygen levels. ARDS can becaused by any major lung inflammation or injury. Some common causesinclude pneumonia, septic shock, trauma, aspiration of vomit, chemicalinhalation and chemotherapy. When a patient is suffering from ARDS,blood concentration of oxygen can remain dangerously low in spite ofsupplemental oxygen delivered by a mechanical ventilator through anendotracheal tube and many will succumb to ARDS. Typically patientsrequire care in an intensive care unit. Symptoms usually develop within24 to 48 hours of the original injury or illness.

Various drugs used for the chemotherapy of cancer damage the lungresulting in severe respiratory toxicities that can lead to ARDS. Forexample, 6 of 151 testicular cancer patients treated with cisplatin (20mg/m²/d IV, days 1-5), etoposide (75 mg/m²/d IV, days 1-5), ifosfamide(1.2 g/m², days 1-5) and mesna (120 mg/m² IV before ifosfamide on day 1,followed by 1.2 g/m² on days 1-5), repeat cycle every 3 weeks for four3-week periods, developed grade III-IV respiratory toxicity. Nichols, J.R., et al., J. Clin. Oncol. 16: 1287-93 (1998). Also, 2 out of 40patients with bladder cancer treated with gemcitabine 1200 mg/m² IV(administered weekly times three on a 4-week cycle) experienced gradeIII-IV respiratory toxicity. Stadler, W. M., et al., J. Clin. Oncol. 15:3394-98 (1997). Moreover, 18% of Non-Hodgkin's lymphoma patients treatedwith cyclophosphamide (600, 750 or 1000 mg/m² IV day 1) and fludarabine(20 mg/m²/d IV over 30 minutes, days 1-5), repeat cycle for 3 or 4weeks, developed grade III or IV pulmonary toxicity including a case ofdocumented pneumocystis carinii pneumonia, leading to discontinuation oftreatment for 11% of patients (3 of 27 patients) because of pulmonarytoxicity. Hochster, H. S. et al., J. Clin. Oncol. 18(5): 897-94 (2000).In addition, 7% of patients with newly diagnosed advanced Hodgkin'sdisease and treated with doxorubicin (25 mg/m² IV days 1, 15), bleomycin(10 mg/m² IV days 1, 15), vinblastine (6 mg/m² IV days 1, 15) anddacarbazine (375 mg/m² IV days 1, 15) developed grade III or IVpulmonary toxicity with a mortality rate of 3% due to pulmonarytoxicity. Canellos, G. P. et al., N. Engl. J. Med. 327(21): 1478-84(1992). Twenty percent of acute promyelocytic leukemia patients treatedwith all-trans-retinoic acid developed severe (7%), life-threatening(11%) or lethal (2%) grade III or IV pulmonary toxicity. Tallman, M. S.et al., N. Eng. J. Med., 337(15): 1021-8 (1997).

Moreover, neutropenia is often associated with cancer chemotherapy. See,for example, Canellos, G. P. et al., N. Engl. J. Med. 327(21): 1478-84(1992); Stadler, W. M., et al., J. Clin. Oncol. 15: 3394-98 (1997);Rose, P. G. et al., J. Clin. Oncol., 17: 2678-80 (1999). Neutropenia isan abnormally low level of neutrophils in the blood and a large body ofclinical data indicates that susceptibility to infectious diseasesincrease sharply when neurophil levels fall below 1000 cells/μL.Holland, S. M. and Gallin, J. I. “Disorders of Granulocytes andMonocytes,” in Harrison's Principles of internal medicine, Vol. 1,Fauci, A. S. et al., eds., 14^(th) ed., McGraw-Hill, New York, N.Y., pp.351-359 (1998). Moreover, control of endogenous microbial flora becomesimpaired when the absolute neutrophil count falls below 500 cell/μL.

Table 1 lists additional toxicity data reported in the scientificliterature for various chemotherapeutic agents used to treat variouscancers.

Leukopenia/ Neutropenia Other Grade III-IV Drug Dose and Target ToxicityToxicities BRAIN CANCER BCNU (carmustine) Leukopenia Thrombocytopenia21% 200 mg/m² IV daily Grade III 4% Nausea/Vomiting 5% Repeat cycleevery 6-8 weeks Grade IV 1% Procarbazine 60 mg/m²/d PO days 8-21Leukopenia Thrombocytopenia 26% Lomustine 110 mg/m² PO day 1 only GradeIII 15% Nausea/Vomiting 11% Vincristine 1.4 mg/m² IV on days 8 and 29Grade IV 8% Neuropathy 8% Repeat cycle every 6-8 weeks Temozolomide 150mg/m²/d PO days 1-5* Neutropenia 2% Fever 2% Repeat cycle every 28 daysLeukopenia 2% Thrombocytopenia 6% *For patients who have previouslyreceived Nausea 10% any chemotherapy. Dose for chemotherapynaiveVomiting 6% patients was 200 mg/m²/d. Headache 6% Asthenia 6% Fatigue 5%Anemia Grade III-IV 1% BREAST CANCER Doxorubicin 60 mg/m² IV day 1 GradeIII 5% Neutropenic Sepsis 3% Cyclophosphamide 600 mg/m² IV day 1 GradeIV 2% Neutropenic Infection 3% Repeat cycle every 21 days Vomiting 13%Nausea 9% Diarrhea <1% Stomatitis 1% Phlebitis 1% Cardiac <1%Cyclophosphamide 500 mg/m² IV day 1 Leukopenia Gastrointestinal 20%Doxorubicin 50 mg/m² IV day 1 Grade III 56% Stomatitis 2% 5-Fluorouracil500 mg/m² IV day 1 Grade IV 25% Alopecia 55% Repeat cycle every 21 days.Cardiac 1% Thrombocytopenia 8% Anemia Grade III-IV 9% Capecitabine 1250mg/m² PO twice daily Grade III-IV 3% Thrombocytopenia 4% days 1-14.Hand-Foot Syndrome Repeat cycle every 21 days. 10% Diarrhea 14% Nausea4% Vomiting 4% Fatigue 7% Stomatitis 3% Dehydration 4% Anemia GradeIII-IV 4% Cyclophosphamide 500 mg/m² IV day 1 Grade III-IV 86%Neutropenic Infection Epirubicin 100 mg/m² IV day 1 (Grade IV) or5-Fluorouracil 500 mg/m² IV day 1 Neutropenic Fever 8% Repeat cycleevery 28 days Thrombocytopenia 6% Nausea/Vomiting 30% Mucositis 10%Alopecia 72% Cardiac Toxicity* 11% *>15% drop from baseline or >10% dropbelow low level of normal Anemia Grade III-IV 7% Docetaxel 100 mg/m² IVover 1 hr every 21 days Grade III-IV 93% Neutropenic Fever 9%Neutropenic Infection (Grade III-IV) 11% Thrombocytopenia 4% Nausea 5%Vomiting 3% Stomatitis 9% Diarrhea 8% Skin Toxicity 4% Asthenia 16%Neurosensory 5% Severe Fluid Retention 8% Docetaxel 75 mg/m² IV day 1Neutropenia Hyperbilirubinemia 8.8% Capecitabine 1250 mg/m² PO twicedaily Grade III 19% Diarrhea 14% days 1-14 Grade IV 44% Stomatitis 17%Repeat cycle every 3 weeks Hand-Foot Syndrome 24% Nausea 6%Fatigue/Asthenia 7% Doxorubicin 50 mg/m² IV day 1 Grade III-IV 97%Febrile Neutropenia 33% Docetaxel 75 mg/m² IV day 1 NeutropenicInfection Repeat cycle every 21 days (Grade III-IV) 8% Allergy 1% Nausea6% Vomiting 6% Stomatitis 9% Diarrhea 8% Asthenia 9% Edema 1% NailChanges 1% Cardiac 6% Doxorubicin 50 mg/m² IV day 1, followed 24Neutropenia Infection (Grade III) 2% hours later by Grade III 27% Fever8% Paclitaxel 220 mg/m² IV over 3 hours day 2. Grade IV 62%Thrombocytopenia Repeat cycle every 21 days. (Grade IV) 2%Arthralgia/Myalgia (Grade III) 10% Peripheral Neuropathy (Grade III) 12%Nausea/Vomiting (Grade III) 8% Diarrhea (Grade III) 2% Stomatitis (GradeIII) 1% Anemia Grade III-IV 9% Paclitaxel 135 mg/m² IV over 3 hrs, day 1Grade III 22% Neutropenic Fever 18% Vinorelbine 30 mg/m² IV days 1, 8Grade IV 71% (Most common with Repeat cycle every 28 days increasedLFTs) Thrombocytopenia 4% Constipation 2% Nausea/Vomiting 2% Alopecia86% Phlebitis 12% Anemia Grade III-IV 12% Paclitaxel 135 mg/m² IV over 3hrs, day 1 Grade III 22% Neutropenic Fever 18% Vinorelbine 30 mg/m² IVdays 1, 8 Grade IV 71% (Most common with Repeat cycle every 28 daysincreased LFTs) Thrombocytopenia 4% Constipation 2% Nausea/Vomiting 2%Alopecia 86% Phlebitis 12% Anemia Grade III-IV 12% Trastuzumab 4 mg/kgload IV followed by Grade III-IV 30% Headache 3% 2 mg/kg IV weekly,followed by Pancreatitis 3% Vinorelbine 25 mg/m² IV weekly Assessmentdone at 8-week intervals Vinorelbine 30 mg/m² IV weekly Grade III-IV 72%Neutropenic Infection <1% Thrombocytopenia 1% Nausea/Vomiting <1%Alopecia 1% Stomatitis <1% Neuropathy 1% Constipation 3% Anemia GradeIII-IV 5% GASTROINTESTINAL: COLON CANCER Capecitabine 1250 mg/m² POtwice daily Leukopenia Thrombocytopenia 1% days 1-14 Grade III 0.3%Diarrhea 15.4% Repeat cycle every 3 weeks Neutropenia Hand-Foot GradeIII 1.3% Syndrome 18.1% Grade IV 1.3% Hyperbilirubinemia 17.3%Stomatitis 3% Hyperglycemia 8.7% Anemia Grade III-IV 1.3% 5-Fluorouracil750 mg/m²/d IV continuous Grade III-IV 0% Diarrhea* 22% infusion, days1-7 Stomatitis* 31% Repeat cycle every 21 days Hand-Foot Syndrome* 14%*Grade II-IV toxicity Leucovorin 20 mg/m²/d IV days 1-5 LeukopeniaNausea* 10% 5-Fluorouracil 425 mg/m²/d IV following Grade III-IVVomiting* 9% leucovorin, days 1-5 21% Diarrhea* 14% Repeat cycle at 4weeks, 8 weeks, then Stomatitis* 26% every 5 weeks thereafter Alopecia34% *Toxicity ungraded, but listed as severe Irinotecan 350 mg/m² IVover 30 minutes Pretreated pts Neutropenic Fever 14% Repeat cycle every21 days Grade III-IV Documented Infection 47% 5% Naïve pts Pre-treatedpatients Grade III-IV (n = 165) 48% Thrombocytopenia 3% Delayed Diarrhea39% Nausea/Vomiting 22% Alopecia 53% Anemia Grade III-IV 10% Naïve pts(n = 48) Thrombocytopenia 2% Delayed Diarrhea 35% Nausea/Vomiting 13%Alopecia 54% Anemia Grade III-IV 8% Irinotecan 180 mg/m² IV day 1 GradeIII-IV 29% Neutropenic Fever 5-Fluorouracil 400 mg/m² IV, followed byWithout Infection 9% 600 mg/m² IV infusion over 22 hours NeutropenicInfection 2% plus leucovorin 200 mg/m² Diarrhea 44% on days 1 and 2Nausea 7% Repeat cycle every 2 weeks Vomiting 11% Asthenia 7% Anorexia7% Abdominal Pain 6% Cholinergic Syndrome 2% Pain 2% Weight Loss 2%Anemia Grade III-IV 6% Irinotecan 125 mg/m² IV over 90 minutesNeutropenia Neutropenic Fever 7.1% days 1, 8, 15, 22, followed by GradeIII 29.8% Neutropenic Leucovorin 20 mg/m² IV Grade IV 24% Infection 1.8%days 1, 8, 15, 22, followed by Diarrhea 22.7% 5-Fluorouracil 500 mg/m²IV Vomiting 9.7% days 1, 8, 15, 22 Mucositis (Grade III) Repeat cycleevery 6 weeks 2.2% GASTROINTESTINAL: COLORECTAL CANCER Irinotecan 125mg/m² IV over 90 minutes Neutropenia Neutropenic Fever 7.1% days 1, 8,15, 22, followed by Grade III 29.8% Neutropenic Leucovorin 20 mg/m² IVGrade IV 24% Infection 1.8% days 1, 8, 15, 22, followed by Diarrhea22.7% 5-Fluorouracil 500 mg/m² IV Vomiting 9.7% days 1, 8, 15, 22Mucositis (Grade III) Repeat cycle every 6 weeks 2.2% GASTROINTESTINAL:ESOPHAGEAL CANCER Cisplatin 100 mg/m² IV day 1 Leukopenia FebrileNeutropenia 5-Fluorouracil 1000 mg/m²/d IV continuous Grade III-IVLeading to Septicemia infusion, days 1-5 14% and Death 5% Repeat cycleevery 21 days Nausea/Vomiting (Grade III) 27% Thrombocytopenia 14%Diarrhea (Grade III) 2% Mucositis (Grade III) 4% Vascular Thrombosis(Grade III) 9% GASTROINTESTINAL: GASTRIC CANCER Etoposide 120 mg/m² IVdays 4, 5, 6 Leukopenia 30% Neutropenic Sepsis 1% Doxorubicin 20 mg/m²IV days 1, 7 Thrombocytopenia 9% Cisplatin 40 mg/m² IV days 2, 8Mucositis 10% Repeat cycle every 21-28 days Alopecia (Grade II-IV) 100%Leucovorin 300 mg/m²/d IV over 10 minutes, Leukopenia NeutropenicInfection 2% followed by Grade III 16% Thrombocytopenia 4% Etoposide 120mg/m²/d IV over 50 minutes, Grade IV 4% Diarrhea 7% followed by Alopecia65% 5-Fluorouracil 500 mg/m²/d IV over 10 minutes Stomatitis All agentsare given on days 1-3 (Grade II-IV) 10% Repeat cycle every 21-28 days5-Fluorouracil 600 mg/m² IV days 1, 8, 29, 36 Leukopenia 3% NeutropenicInfection Doxorubicin 30 mg/m² IV days 1, 29 20% Mitomycin 10 mg/m² IVday 1 Infection (any)* 20% Repeat cycle every 8 weeks Nausea/Vomiting*58% Thrombocytopenia 5% Mucositis* 10% Diarrhea* 28% Alopecia* 57%*Grade II-IV toxicity or unspecified Anemia Grade III-IV 3% Methotrexate1500 mg/m² IV day 1 Neutropenic Sepsis 2% 5-Fluorouracil 1500 mg/m² IVstarting 1 hour Nausea/Vomiting 8% after methotrexate, day 1 Mucositis10% Leucovorin 15 mg/m² PO q6h starting Diarrhea* 26% 24 hours aftermethotrexate dose, Alopecia 24% for 48 hours *Unspecified gradeDoxorubicin 30 mg/m² IV day 15 of toxicity Repeat cycle every 28 daysGASTROINTESTINAL: LIVER (HEPATOCELLULAR) CANCER Gemcitabine 1250 mg/m²IV Leukopenia Infection over 30 minutes days 1, 8, 15 Grade III (GradeIII) 3.6% Repeat cycle every 28 days 10.7% Thrombocytopenia 10.7%Hepatotoxicity (Grade III) 14.3% Skin Rash (Grade III) 3.6% Anemia GradeIII 14.3% GASTROINTESTINAL: PANCREATIC CANCER 5-Fluorouracil 600 mg/m²IV days 1, 8, 29, 36 Leukopenia Thrombocytopenia 30% Doxorubicin 30mg/m² IV days 1, 29 Grade III 16% Hepatic 1% Mitomycin 10 mg/m² IV day 1Grade IV 6% Renal 2% Repeat cycle every 72 days Nausea/Vomiting 11%Cardiac 2% Gemcitabine 1000 mg/m² IV over 30 minutes Grade III 19%Thrombocytopenia 10% once weekly for 7 weeks, followed by a Grade IV 7%Increased Bilirubin 41% 1-week rest period Nausea/Vomiting 13%Subsequent cycles once weekly for 3 Diarrhea 2% consecutive weeks out ofevery 4 weeks Constipation 3% Pain 2% State of Consciousness 2% AnemiaGrade III-IV 10% GASTROINTESTINAL: RECTAL CANCER Leucovorin 500 mg/m² IVover 2 hours, Leukopenia Infection (any) 14% 1 hour prior to5-fluorouracil, every week × <4 × 103/mm³ (systemic +/or sepsis) 3% 6weeks, then 2 weeks off 65% Fever (any) 9% (>40° C. 5-Fluorouracil 500mg/m² IV bolus Leukopenia or hypotension) 1% every week × 6 weeks, then2 weeks off <2 × 103/mm³ Thrombocytopenia 5-Fluorouracil 400 mg/m² IVbolus daily 3% <100K 4% on days 1-3 and the last 3 days ofNausea/Vomiting (any) radiation therapy 59% Duration of each cycle: 10weeks (severe or with 5-Fluorouracil/leucovorin for a total of 6 cycleshospitalization) 4% Radiation therapy initiated between 3 and Diarrhea(≧3 stools/day) 5 weeks following completion of cycle 1 61% (≧7stools/day) 31% Stomatitis (any) 21% Dermatitis (any) 25% (severe) 3%GENITOURINARY: BLADDER CANCER Cisplatin 30 mg/m² IV Grade III-IV 8%Neutropenic Fever 8% days 1, 8, 15, 22, 29, 36, 43, 50 Nausea/Vomiting8% Docetaxel 40 mg/m² IV Diarrhea 35% days 4, 11, 18, 25, 32, 39, 46, 53Abdominal Pain 35% Urinary Frequency 19% Dysuria 14% Severe Cystitis 8%Thrombocytopenia 11% Anemia Grade III-IV 16% Paclitaxel 175 mg/m² IVover 3 hours day 1, Granulocytopenia Neutropenic Fever 1.9% followed byGrade III-IV Infection Cisplatin 75 mg/m² IV over 1-2 hours day 1 52%(non-neutropenic) 9.6% after paclitaxel Nausea/Vomiting 29% Repeat cycleevery 21 days Neurosensory 15.4% Neuromotor 11.5% Metabolic 17.3%Cardiac 7.7% Pulmonary 3.8% Neuropsychological/ Neuroclinical 7.7%Hepatotoxicity 3.8% Anemia Grade III 9.6% Gemcitabine 1000 mg/m² IV days1, 8, 15 Neutropenia Neutropenic Fever 2% Cisplatin 70 mg/m² IV day 2Grade III Neutropenic Sepsis 1% Repeat cycle every 28 days 41.2%Thrombocytopenia 57% Grade IV Nausea/Vomiting 29.9% (Grade III) 22%Alopecia (Grade III) 10.5% Diarrhea (Grade III) 3% Hematuria (Grade III)4.5% Pulmonary 3% Anemia Grade III-IV 27% Gemcitabine 1200 mg/m² IV days1, 8, 15 Grade III-IV 20% Neutropenic Fever 3% Repeat cycle every 28days Thrombocytopenia 5% Nausea/Vomiting 5% Fever 3% Edema 5% Deep VeinThrombosis 3% Respiratory 5% CNS 3% Anemia Grade III-IV 3% Methotrexate30 mg/m² IV days 1, 15, 22 Leukopenia Neutropenia Vinblastine 3 mg/m² IVdays 2, 15, 22 Grade III 38% Nadir Sepsis 25% Doxorubicin 30 mg/m² IVday 2 Grade IV 20 Nausea/Vomiting 7% Cisplatin 70 mg/m² IV day 2Thrombocytopenia 21% Repeat cycle every 28 days Mucositis 13% Renal 2%Hepatic 5% Diarrhea 2% Paclitaxel 250 mg/m² IV over 24 hours, day 1Grade III-IV 23% Neutropenic Fever* 8% Repeat cycle every 21 days Allpatients Neuropathy 12% received G-CSF Mucositis 12% Diarrhea (Grade IV)4% *All pts received G-CSF Paclitaxel 200 mg/m² IV over 1 hour on day 1Leukopenia Thrombocytopenia Gemcitabine 1000 mg/m² IV Grade III 37%(Grade III) 13% over 30 minutes on days 1, 8, and 15 Grade IV 9%Fatigue/Asthenia 11% Repeat cycle every 21 days Peripheral Neuropathy11% Nausea/Vomiting 7% Arthralgia/Myalgia 6% Skin Rash 6%Hypersensitivity Reaction 4% Pneumonitis 2% Anemia Grade III 28%Paclitaxel 225 mg/m² over 3 hours on day 1 Grade III-IV 41% NeutropenicFever 12% Carboplatin targeted by Calvert equation Thrombocytopenia 4%to AUC of 6 IV day 1 after paclitaxel Nausea/Vomiting 12% Repeat cycleevery 21 days Neuropathy 20% GENITOURINARY: PROSTATE CANCER Estramustine10 mg/kg/d PO (3 divided Leukopenia Infection doses) days 1-5 on anempty stomach Grade III 57% (site unknown) 24% Docetaxel 70 mg/m² IVover 1 hour day 2 Grade IV 4% Thrombocytopenia 20% Hydrocortisone 30 mgPO every morning and Neutropenia Hyperglycemia 18% 10 mg every eveningcontinuously Grade III 26% Hypocalcemia Repeat estramustine/docetaxelcycles Grade IV 30% (Grade III) 2% every 21 days. Phlebitis/Thrombosis6% Edema 22% Malaise/Fatigue/ Asthenia 24% Stomatitis/Esophagitis (GradeIII) 6% Nausea (Grade III) 4% Vomiting (Grade IV) 2% Diarrhea 6%Anorexia 4% Hepatic 22% Dyspnea 22% Dysrhythmias (Grade III) 2% Ischemia(Grade III) 7% BUN/SCr (Grade III) 9% Renal Failure (Grade IV) 2%Neurologic (Grade III) 8% Estramustine 200 mg/m² PO tid days 1-42 GradeIII 7% Neutropenic Infection 7% Vinblastine 4 mg/m² IV weekly for 6weeks, Grade IV 1% Thrombocytopenia* 1% begin day 1 Nausea* 28% Repeatcycle every 8 weeks Leg Edema* 12% Fatigue* 16% Neurologic* 12%Constipation* 3% Cardiac* 5% *Grade II-IV toxicity Mitoxantrone 12 mg/m²IV day 1 Grade III-IV 45% Neutropenic Sepsis 1% Prednisone 5 mg PO bidcontinuously Thrombocytopenia 5% Repeat cycle every 21 days Cardiac 4%Nausea/Vomiting <1% Alopecia 24% Paclitaxel 30 mg/m²/d IV continuousLeukopenia Neutropenic Fever 6% infusion days 1-4 Grade III-IV Edema 15%Estramustine 600 mg PO daily 24 hr before 21% Cardiovascular 6%paclitaxel (2 to 3 divided doses) Anorexia 21% Repeat cycle every 21days Fatigue 9% Nausea 6% Hepatic 9% Venous Thrombosis 3% Vomiting 3%Diarrhea 3% Vinorelbine 25 mg/m² IV days 1, 8 Leukopenia Infection 4%Estramustine 140 mg PO three times a day Grade III 4% Deep VeinThrombosis days 1-14 4% Repeat cycle every 3 weeks Myocardial Ischemia4% Fatigue 4% Hearing Loss 4% Dyspnea 4% GENITOURINARY: RENAL CELLCANCER Interferon alfa-2a 3 MU SQ daily, Neutropenia Thrombocytopeniaescalated by 3 MU increments every Grade III 3% (Grade III) 1% 7 days,as tolerated to a maximum dose Leukopenia Gastrointestinal of 9 MU SQdaily Grade III 7% (Grade III) 13% Altered Mood (Grade III) 3%Neurologic Toxicity 3% Cardiac Toxicity 5% Anemia Grade III-IV 11%Interferon alfa-2a 18 MU SQ Leukopenia 0.7% Fever 5% TIW × 10 weeks(induction) then Nausea/Vomiting 5% ×13 additional weeks (maintenance)Pulmonary Symptoms 3% Increased AST/ALT 3% Weight Loss 6% Anemia GradeIII-IV 6% Interleukin-2 18 MU/m²/d as an IV Leukopenia 2% Infection 9%continuous infusion × 5 days Fever 56% Interferon alfa-2a 6 MU SQ TIWHypotension Resistant Treatment consists of 2 induction cycles toVasopressors 67% and 4 maintenance cycles, with a Nausea/Vomiting 31%3-week rest period between cycles. Diarrhea 25% An induction cycleconsists of two 5-day Pulmonary Symptoms courses, separated by a 6-dayrest period. 15% Interferon-alfa is given during the two Renal Symptoms16% induction cycles and during each Neurologic Symptoms maintenancecycle. 14% Increased AST/ALT 11% Cutaneous Signs 14% Cardiac Signs 6%Thrombocytopenia 7% Increased Creatinine 5% Hyperbilirubinemia 2% AnemiaGrade III-IV 16% Interleukin-2 600,000 or 720,000 IU/kg IV None reportedSepsis 6% over 15 minutes every 8 hours × 14 doses Fever and/or Chills24% Repeat after a 9-day rest period. Oliguria/Anuria 46% Mental StatusChanges 28% Nausea/Vomiting 25% Diarrhea 22% Hyperbilirubinemia 21%Thrombocytopenia 21% Dyspnea 17% Hypotension 74% Elevated BUN/Creatinine 14% Increased AST/ALT 10% Cardiac Toxicity 9% IncreasedAlkaline Phosphatase 8% Acidosis 6% Asthenia 4% Pruritus 4% Stomatitis4% Gastrointestinal Bleeding 4% Anemia Grade III-IV 18% GENITOURINARY:TESTICULAR CANCER Bleomycin 30 units IV days 2, 9, 16 HematologicNeutropenic Infection 5% Etoposide 100 mg/m²/d IV days 1-5 Grade III 39%Nausea/Vomiting 70% Cisplatin 20 mg/m²/d IV days 1-5 Grade IV 34%Neurologic 7% Repeat cycle every 21 days × 4 Respiratory 5% Hepatic 3%Pulm. Hemorrhage <1% Resp. Failure 1% Etoposide 100 mg/m²/d IV days 1-5At day 21 24% Neutropenic Nadir Cisplatin 20 mg/m²/d IV days 1-5 Sepsis10% Repeat cycle every 21 days Nausea/Vomiting* 44% Stomatitis 2%Alopecia† ~100% *Grade I-II toxicity †Unknown grade of toxicityCisplatin 20 mg/m²/d IV days 1-5 Grade III-IV 59% Neutropenic Sepsis 4%Vinblastine 0.15 mg/kg IV days 1, 2 Thrombocytopenia 5% Bleomycin 30units IV days 2, 9, 16 Paresthesias* 38% Repeat cycle every 21 daysAbdominal Pain* 20% Myalgias* 19% *Unknown grade Vinblastine 0.11 mg/kgIV days 1, 2 Toxicity data reported Ifosfamide 1200 mg/m²/d IV days 1-5on 17 patients: (with mesna uroprotection) 89% neutropenic feverCisplatin 20 mg/m²/d IV days 1-5 requiring antibiotics Repeat cycleevery 21 days 59% required RBC transfusions 35% required platelettransfusions 1 therapy-related death due to CNS toxicity Etoposide 75mg/m²/d IV days 1-5 Hematologic Neutropenic Infection 6% Ifosfamide 1200mg/m²/d IV days 1-5 Grade III 60% Nausea/Vomiting 9% Cisplatin 20mg/m²/d IV days 1-5 Neurological 8% Mesna 120 mg/m² IV 15 minutes priorto Respiratory 40% ifosfamide, then 1200 mg/d IV continuous Hepatic 3%infusion days 1-5 Renal 5% Repeat cycle every 21 days GYNECOLOGIC:CERVICAL CANCER Cisplatin 100 mg/m² IV day 1 Leukopenia Thrombocytopenia1% Repeat cycle every 21 days Grade III-IV 7% Nephrotoxicity (SCr >2.0or BUN >40) 14% Nausea/Vomiting* 74%-83% Hearing Loss (clinical) 12%Neurotoxicity 7% *Toxicity grading unknown Radiation therapy 1.7 Gy/d onLeukopenia Infection (Grade III) days 1-5 of each week, for a total ofGrade III 33% 0.8% 29 fractions (49.3 Gy) Grade IV 2.5% Diarrhea 9.8%Cisplatin 70 mg/m² IV over 2 hours day 1 Granulocytopenia Nausea (GradeIII) 14% 5-Fluorouracil 1000 mg/m²/d IV continuous Grade III 19.6% SmallBowel Obstruction infusion days 1-4 Grade IV 9% (Grade IV) 1.6% 2ndcycle began on day 22, the 3rd and 4th Stomatitis 2.4% chemotherapycycles scheduled after completion Vomiting 12.3% of RT, to begin on days43 and 64 Anemia Grade III-IV 3.3% Cisplatin 50 mg/m² IV days 1, 29Leukopenia Thrombocytopenia 4% 5-Fluorouracil 1000 mg/m²/d IV continuousGrade III 41% Gastrointestinal 18% infusion days 1-4 and 29-32 Grade IV5% Genitourinary 2% Hydroxyurea 2000 mg/m² PO Cutaneous 5% twice weeklytwo hours before Neurologic 1% radiotherapy at weeks 1-6 Cardiovascular2% Fatigue 1% Pain 1% Weight Loss 2% Cisplatin 80 mg/m² IV day 1 GradeIII* 52% Neutropenic Fever 0% Vinorelbine 25 mg/m² IV days 1, 8 GradeIV* 4% Nausea/Vomiting 6% Repeat cycle every 21 days *Per cyclePeripheral Neuropathy 2% Alopecia 4% Anemia Grade III-IV 10% Cisplatin40 mg/m² IV each week Leukopenia Gastrointestinal 14% during XRT (not toexceed 70 mg/wk) × Grade III 18% 6 doses maximum Grade IV 3% XRT 1.8-2.0Gy daily, 5 days per week for a total of 45 Gy, followed by localbrachytherapy and hysterectomy Paclitaxel 135 mg/m² IV over 24 hoursGrade III 16% Neutropenic Fever 28% day 1, followed by Grade IV 61%Fever 5% Cisplatin 75 mg/m² IV day 2 Thrombocytopenia 18% Repeat cycleevery 21 days. Gastrointestinal 36% Neurologic 7% Cardiac 7% Pulmonary7% Dehydration 2% Electrolyte Abnormalities 2% Pneumonia 2% Anemia GradeIII-IV 25% GYNECOLOGIC: ENDOMETRIAL CANCER Doxorubicin 60 mg/m² IV day 1Leukopenia Thrombocytopenia 20% Cisplatin 60 mg/m² IV day 1 given 12hours Grade III 17% Nausea/Vomiting* 53% after doxorubicin Grade IV 43%*Mild to severe Repeat cycle every 28 days anemia Grade III-IV (Hgb <10g) 63% Doxorubicin 60 mg/m² IV day 1 Grade III-IV 8% Gastrointestinal 3%Repeat cycle every 21 days GYNECOLOGIC: OVARIAN CANCER Altretamine 260mg/m²/d PO Nausea 14% (divided into 3 or 4 doses) days 1-14 Vomiting 12%Repeat cycle every 28 days Fatigue 9% Neurologic 2% Anemia Grade III-IV2% Paclitaxel 175 mg/m² IV over 3 hrs, Grade III 31% Nausea/Vomiting 16%day 1, followed by Grade IV 45% Neurotoxicity 1% Carboplatin dosetargeted by the Calvert Thrombocytopenia 4% equation to AUC of 5, IV day1 Repeat cycle every 21 days. Cisplatin 100 mg/m² IV day 1 Grade III 36%Neutropenic Fever <1% Repeat cycle every 21 days Grade IV 12%Thrombocytopenia 4% Gastrointestinal 33% Renal 4% Neurologic 11%Cardiovascular 2% Anemia Grade III-IV 11% Paclitaxel 135 mg/m² IVcontinuous infusion Neutropenia Grade Infection 2% over 24 hours day 1,followed by III 49% Fever 2% Cisplatin 75 mg/m² IV day 2 Grade IV 13%Thrombocytopenia 3% Repeat cycle every 21 days. Other Hematologic 88%Gastrointestinal 17% Cardiovascular 3% Neurologic 9% Metabolic 2%Creatinine Clearance 2% Allergic Reaction 2% Fatigue (Grade III) 1% n =32 Docetaxel 100 mg/m² IV over 1 hour day 1 Granulocytopenia FebrileNeutropenia 33% Repeat cycle every 21 days. Grade III 6%Thrombocytopenia Grade IV 72% (Grade III) 6% anemia Grade III 32%Docetaxel 75 mg/m² IV over 1 hour day 1, Neutropenia Febrile Neutropeniafollowed by Grade III 9% (Grade IV) 3% Carboplatin AUC of 5 (via CrEDTA)or Grade IV 77% Neutropenia >7 days AUC of 6 (if CrCl calculated usingCockroft Leukopenia (Grade IV) 14% and Gault formula) IV day 1 Grade III59% Thrombocytopenia 14% Repeat cycle every 21 days. Grade IV 5% Nausea(Grade III) 5% Vomiting (Grade III) 5% Fatigue (Grade III) 5% AnemiaGrade III-IV 14% Liposomal doxorubicin 50 mg/m² IV day 1 Grade III-IV20% Neutropenic Fever 11% Repeat cycle every 21 days Stomatitis 14%Palmar-Plantar Erythrodysesthesia 29% Paclitaxel 135 mg/m² IV LeukopeniaNeutropenic Fever 33% over 3 or 24 hrs, day 1 Grade III-IV Infection 12%Repeat cycle every 21 days. 78% Cardiac 2% Thrombocytopenia 8% Vomiting7% Mucositis 1% Neurologic 2% Topotecan 1.5 mg/m²/d IV Grade III 15%Neutropenic Fever 18% over 30 min, days 1-5 Grade IV 82% (after theseadverse Repeat cycle every 21 days effects, G-CSF was given) Sepsis 9%Nausea 7% Vomiting 4% Fatigue 5% Thrombocytopenia 52% Severe Bleed <1%Anemia Grade III-IV 49% GYNECOLOGIC: UTERINE SARCOMA Ifosfamide 1500mg/m²/d IV days 1-5 Neutropenia Thrombocytopenia 5% Mesna uroprotectionat equivalent doses to Grade III 28% Nausea/Vomiting ifosfamide via IVcontinuous infusion Grade IV 31% (Grade III) 4% Repeat cycle every 21days Granulocytopenia Other Gastrointestinal Grade III 8% (Grade IV) 1%Grade IV 28% Creatinine Elevation (Grade III) 2% Neurologic-Central(Grade III) 19% Neurologic-Peripheral (Grade IV) 1% Hematuria (GradeIII) 1% Anemia Grade III-IV 8% HEAD & NECK CANCER Carboplatin 300 mg/m²IV day 1 Leukopenia Neutropenic Sepsis 1% 5-Fluorouracil 1000 mg/m²/d IVcontinuous Grade III 9% Nausea/Vomiting 6% infusion days 1-5 Grade IV 2%Thrombocytopenia 13% Repeat cycle every 28 days Diarrhea 2% Stomatitis15% Renal Toxicity 1% Anemia Grade III-IV 14% Paclitaxel 175 mg/m² IVover 3 hours day 1 Neutropenia Neutropenic Fever Ifosfamide 1000 mg/m²/dIV over 2 hours Grade III-IV (Grade V*) 2% days 1-3 92% Fatigue (GradeIII) 6% Mesna 200 mg/m² before ifosfamide and Nausea/Emesis 400 mg/m²after ifosfamide days 1-3 (Grade III) 4% Carboplatin targeted to an AUCof 6 (Calvert Peripheral Sensory formula) as a 30-minute IV infusion day1 Neuropathy Repeat cycle every 21-28 days. (Grade III) 2%Myalgia/Arthralgia (Grade III) 2% Nephrotoxicity (Grade III) 2% Anorexia(Grade III) 4% *NCIC criteria used, scale Grade 0-V Paclitaxel 175 mg/m²over 3 hours on day 1 Grade III-IV 90% Neutropenic Fever 27% Ifosfamide1000 mg/m²/d over 2 hours on Pneumonia 15% days 1-3 Nausea/Vomiting 6%Mesna 400 mg/m² IV before ifosfamide Anorexia 4% and 200 mg/m² IV, 4 hrafter ifosfamide Fatigue 15% days 1-3 Peripheral Neuropathy Cisplatin 60mg/m² IV day 1 6% Repeat cycle every 21-28 days. Renal Toxicity 2%Mucositis 2% Orthostatic Hypotension 4% LEUKEMIAS: ACUTE LYMPHOBLASTIC,ADULT Methotrexate 200 mg/m² IV over 2 hours Sepsis 11% day 1, followedby: Pneumonia 5% Methotrexate 800 mg/m² IV over 24 hours FungalInfections <1% day 1 Fever of Unknown Origin Calcium leucovorin 15 mg POevery 6 hours × 23% 8 doses starting 24 hours after completion Rash 5%of the MTX infusion-dose adjusted based Mucositis 5% on MTX levelsNeurotoxicity 5% Cytarabine 3000 mg/m² IV over 2 hours every Rash andDesquamation 12 hours on days 2 and 3 (4 doses total) of Palms/Feet 3%Methylprednisolone 50 mg IV twice daily Diarrhea 1% days 1-3Cytarabine-Associated G-CSF 5 mcg/kg SQ twice daily starting day 4 Fever6% Nephrotoxicity until ANC recovery (reversible) 1.5% Cycle repeatedwhen ANC >3 × 109/L and Cytarabine platelets >60 × 109/L (approximatelyNeurotoxicity 2% 3-4 weeks) Cyclophosphamide 300 mg/m² IV over Time toInduction 3 hours every 12 hours days 1-3 granulocytes Moderate-Severe(total 6 doses) >1 × 109/L: Mucositis 6% Mesna 600 mg/m²/d IV continuousinfusion 18 days after Moderate-Severe days 1-3-continue until 6 hoursafter the first cycle Diarrhea 3% last dose of cyclophosphamideDisseminated Vincristine 2 mg IV days 4, 11 Intravascular Doxorubicin 50mg/m² IV day 4 Coagulopathy Requiring Dexamethasone 40 mg PO dailyTherapy 2% days 1-4 and days 11-14 Consolidation G-CSF 5 mcg/kg SQ twicedaily starting day Neurotoxicity 8% 24 hours after the completion ofchemotherapy G-CSF-Induced Bone and continuing until ANC recovery Pain5% Alternate cycles of Hyper-CVAD with HD MTX-Ara-C every 3-4 weeks(when WBC >3 × 109/L and platelet count >60 × 109/L) LEUKEMIAS: ACUTEMYELOGENOUS Fludarabine 30 mg/m²/d IV over 30 minutes NeutropeniaDocumented Infections days 1-5 Grade IV 100% (>Grade III) 44% Cytarabine2000 mg/m²/d IV over 4 hours Bacterial Infections 34% after completionof fludarabine days 1-5 Fungal Infections 10% G-CSF 5 mcg/kg/d IV day 0Fever of Unknown Origin (24 hours prior to starting chemotherapy) 44%until ANC recovery Mucositis (>Grade III) 10% Diarrhea (>Grade III) 8%Hepatic (>Grade III) 8% Neurologic (>Grade III) 2% Gemtuzumab 9 mg/m² IVover 2 hours Neutropenia Neutropenic Fever 7% Recommended treatmentcourse is a total of Grade III-IV Sepsis 16% 2 doses with 14 daysbetween the doses 98% Fever 15% Leukopenia Chills 13% Grade III-IVThrombocytopenia 99% 96% Dyspnea 9% Pneumonia 7% Nausea 9%Hyperbilirubinemia 23% Hyperglycemia 12% Anemia Grade III-IV 47%Idarubicin 12 mg/m²/d IV days 1-3 Nausea/Vomiting 6% Cytarabine 100mg/m²/d IV continuous Diarrhea 16% infusion days 1-7 Mucositis 7% Onlyrepeat if patient fails to achieve Hyperbilirubinemia 9% a remissionSkin Rash 5% Alopecia 40% Cardiac Toxicity 11% LEUKEMIAS: ACUTEMYELOGENOUS, INDUCTION Cytarabine 100 mg/m²/d IV continuous NeutropenicInfection infusion days 1-5 51% Daunorubicin 45 mg/m²/d IV days 1, 2Hemorrhage 20% *For reinduction Hepatic 7% Renal 5% Cardiac 4%Cytarabine 100 mg/m²/d IV continuous Neutropenic Infection infusion days1-7 54% Daunorubicin 45 mg/m²/d IV days 1-3 Hemorrhage 12% Hepatic 2%Renal 4% Gastrointestinal 1% Etoposide 200 mg/m²/d IV continuous 100%Neutropenic Infection infusion days 8-10 54% Mitoxantrone 12 mg/m²/d IVdays 1-3 Thrombocytopenia 100% Cytarabine 500 mg/m²/d IV continuousMucositis 23% infusion days 1-3, 8-10 Hyperbilirubinemia 8% Bleeding 6%Rash 5% Diarrhea 3% Metabolic Disorders 2% Cerebellar Syndrome 1%Vomiting 9% LEUKEMIAS: ACUTE MYELOGENOUS, POSTREMISSION Cytarabine 100mg/m²/d IV continuous Grade III-IV Patients Requiring infusion days1-5* >16% Hospitalization Due to Repeat cycle every 28 days NeutropenicFever 16% *For patients >60 years of age Patients Requiring TransfusionsDue to Thrombocytopenia 28% Cytarabine 3000 mg/m² IV over 3 hours GradeIII-IV 71% Neutropenic Fever 71% every 12 hours days 1, 3, 5Thrombocytopenia Administer with saline, methylcellulose, or (Grade IV)86% steroid eye drops OU, every 2-4 hours, CNS Toxicities 12% beginningwith cytarabine and continuing (32% with age >60 yrs) 48-72 hours afterthe last cytarabine dose Repeat cycle every 28 days LEUKEMIAS: ACUTEMYELOGENOUS, SALVAGE Cyclophosphamide 1000 mg/m²/d IV over NeutropenicFever 76% 2 hours days 1-3 Hyperbilirubinemia Etoposide 200 mg/m²/d IVover 3 hours (Grade IV) 4% days 1-3 Hemorrhagic Cystitis Carboplatin 150mg/m²/d for 3 days IV (Grade IV) 4% continuous infusion days 1-3Diarrhea (Grade III) 4% Cytarabine 1000 mg/m²/d IV over 2 hoursMucositis (Grade III) 4% days 1-3 Cardiac Toxicity (Grade III) 4%LEUKEMIAS: ACUTE PROMYELOCYTIC Induction Therapy Neutropenia FebrileNeutropenia 8% Arsenic trioxide 0.15 mg/kg/d IV over Grade III-IV Fever5% 1-2 hours from day 1 until bone marrow 10% Disseminated remission(not greater than 60 doses) Intravascular Coagulation ConsolidationTherapy 8% Should begin 3-6 weeks after completion Thrombocytopenia 12%of induction therapy at a dose of Arthralgia/Mylagia 13% 0.15 mg/kg/dfor 25 doses over Dyspnea 10% a period of up to 5 weeks Sepsis 5%Hypoxia 10% Hypokalemia 13% Hypomagnesemia 13% Hyperglycemia 13%Paresthesias 5% Convulsions 5% Leukocytosis 3% Anemia Grade III-IV 5%Induction Induction Therapy Cytarabine 100 mg/m²/d IV continuous Severe= S infusion days 1-7 Life-Threatening = LT Daunorubicin 45 mg/m²/d IVdays 1-3 Lethal = L Consolidation Infection First cycle-identical toinduction S = 38%, LT = 8%, L = 6% Second cycle (postremission)- HepaticS = 13%, LT = 9% Cytarabine 2000 mg/m² IV over 1 hour Hemorrhage every12 hours days 1-4 S = 6%, LT = 4%, L = 7% Daunorubicin 45 mg/m² IV days1, 2 Pulmonary Maintenance therapy-randomized S = 2%, LT = 2%, L = 1%ATRA 45 mg/m²/d PO in divided doses Stomatitis S = 5%, LT = 1% every 12hours for 1 year or Cardiac S = 9% to observation Nausea S = 9% DiarrheaS = 10%, LT = 1% Dermatologic S = 5%, LT = 1% Neurologic S = 7%, LT = 1%Maintenance Therapy (n = 94) Infection 7.4% Life-Threatening Toxicity36% Neurotoxicity 11.7% Hepatotoxicity 5.3% LEUKEMIAS: CHRONICLYMPHOCYTIC Alemtuzumab 30 mg IV over 2 hours three Neutropenia Sepsis10% times a week (initiate dosing at 3 mg IV Grade III-IV Fever 19%daily; when tolerated [infusion-related 64% Thrombocytopenia 50%toxicities are <Grade 2] increase to 10 mg). Fatigue 5% When the 10 mgdose is tolerated, the Rigors 16% maintenance dose of alemtuzumab 30 mgHypotension 5% can be initiated. Maintain at 30 mg TIWBronchitis/Pneumonitis for up to 12 weeks. 13% Pneumonia 10% Urticaria5% Dyspnea 9% Anemia Grade III-IV 38% Chlorambucil 30 mg/m² PO day 1Hematologic Neutropenic Infection 7% Prednisone 80 mg/d PO days 1-5Grade III-IV Neutropenic Fever 2% Repeat cycle every 14 days 25%Vomiting 3% Cladribine 0.12 mg/kg/d IV Granulocytopenia FUO/Infection*56% over 2 hours days 1-5 9% Thrombocytopenia 9% Prednisone 30 mg/m²/dPO days 1-5 Autoimmune Hemolytic Repeat cycle every 28 days Anemia* 6%Hepatic* 6% Nausea/Vomitng* 2% Diarrhea* 2% Anemia Grade III-IV 2%*Grade Unknown Fludarabine 25-30 mg/m²/d IV days 1-5 Neutropenia 56%Febrile Infection During Repeat cycle every 28 days Grade IV Cycles 1-330% (of cycles) Thrombocytopenia 25% Nausea 3% Stomatitis 2% Diarrhea 2%Neuropathy 4% LEUKEMIAS: CHRONIC MYELOGENOUS Interferon 5 MU/m² SQ dailyNausea/Vomiting/ Cytarabine 20 mg/m²/d SQ days 1-10 Diarrhea 12.5% everymonth Mucositis 5.8% Notes: Other GI 1% Decrease dose by 50% forgranulocyte Weight Loss/ count <1500/mm³ and/or platelets Asthenia 13.3%<100,000/mm³ Skin Rash 5.2% Cytarabine held for WBC <3000/mm³ and Fever,Flu-like platelets <100,000/mm³; cytarabine Syndrome 3% discontinued forgranulocyte count Peripheral Neuropathy <1000/mm³ and platelets<50,000/mm³ 1% If hematologic control not achieved, then CentralNeurologic increase cytarabine to 40 mg/m²/d SQ Symptoms 1% days 1-15.Depression 4.2% Other Psychologic 3.6% Cytolytic Hepatitis 2.5% OtherToxicity 8.6% Thrombocytopenia 5.5% LEUKEMIAS: HAIRY CELL Cladribine 0.1mg/kg/d IV continuous Grade III-IV 87% Neutropenic Fever 42% infusiondays 1-7 Documented Administer one cycle Infections 13% Thrombocytopenia20% Anemia Grade III-IV 22% Carboplatin AUC of 6 given IV day 1Leukopenia Thrombocytopenia Paclitaxel 225 mg/m² IV over 3 hours day 1Grade III 26% (Grade III) 10% Repeat cycle every 21 days. Grade IV 5%Nausea (Grade III) 7% Neutropenia Neuropathy (Grade III) Grade III 21%13% Grade IV 36% Vomiting (Grade III) 4% Dehydration (Grade III) 4%Fatigue (Grade III) 8% Anemia Grade III-IV 13% Paclitaxel 215 mg/m² IVover 24 hours, Grade III 26% Febrile Neutropenia 13% day 1, followed byGrade IV 45% Hospitalization (for Carboplatin dose targeted by (G-CSFuse IV antibiotics) 13% Calvert equation to AUC of 7.5 IV after cycle 2)Nausea/Vomiting 8% Repeat cycle every 21 days. Thrombocytopenia 48%Fatigue 21% Anorexia 23% Myalgia/Arthralgia 6% Paresthesia 2% AnemiaGrade III-IV 34% Cisplatin 100 mg/m² IV day 1 LeukopeniaThrombocytopenia Vinorelbine 30 mg/m² IV days 1, 8, 15, 22 Grade III 35%(Grade III) 4% Repeat cycle every 28 days Grade IV 15% Nausea (GradeIII) 18% Neutropenia Neuropathy (Grade III) Grade III 27% 3% Grade IV49% Vomiting (Grade III) 12% Dehydration (Grade III) 6% Fatigue (GradeIII) 11% Hyponatremia (Grade III) 7% Respiratory Infection (withneutropenia) (Grade III) 5% anemia Grade III 17% Docetaxel 36 mg/m² IVLeukopenia Fatigue/Asthenia 10% over 1 hour days 1, 8, 15, 22, 29, 36Grade III 8% Nausea/Emesis 10% Repeat cycle every 8 weeks Skin Toxicity3% Neuropathy 3% Hypersensitivity Reactions 3% Alopecia 13% Anemia GradeIII 13% Docetaxel 75 mg/m² IV day 1 Grade III-IV 67% Hospitalized forFebrile Cisplatin 100 mg/m² IV day 1 Neutropenia 16% Repeat cycle every21 days. Neutropenic Infection 14% Thrombocytopenia 2% Allergy 2%Cardiac Dysrhythmia 4% Diarrhea 6% Nausea 14% Vomiting 14% Alopecia* 12%Asthenia 35% Nail Disorder 4% Neuromotor 2% Neurosensory 2% Anemia GradeIII-IV 10% *Grade II toxicity Etoposide 100 mg/m²/d IV days 1-3Leukopenia Neutropenic Infection* Cisplatin 120 mg/m² IV day 1 GradeIII-IV 34% 5% Repeat cycle every 21-28 days Thrombocytopenia* 15%Nausea/Vomiting* 77% Diarrhea* 11% Bleeding* 3% Polyneuropathy 2% RenalDysfunction* 10% Hypoacusia* 7% Alopecia* 60% *Grade II-IV toxicityGemcitabine 1200 mg/m² IV days 1, 8 Neutropenia ThrombocytopeniaVinorelbine 30 mg/m² IV days 1, 8 Grade III 30% 13.3% Repeat cycle every21 days Grade IV 8.3% Vomiting 15% Fatigue (Grade III) 5% Anemia GradeIII 6.7% Gemcitabine 1000 mg/m² IV over Granulocytopenia NeutropenicFever 4.6% 30-60 minutes days 1, 8, 15, followed by Grade III 21.7%Thrombocytopenia Cisplatin 100 mg/m² IV over Grade IV 35.3% 50.4% 30-120minutes day 1 only Nausea 27% Repeat cycle every 28 days Vomiting 23%Neurohearing 6% Neuromotor (Grade III) 11.5% Cardiac Dysrrhythmia 2.8%Dyspnea 7% Elevated Creatinine 4.8% Anemia Grade III-IV 25% Paclitaxel200 mg/m² IV over 3 hours day 1 Leukopenia Infection 10% Repeat cycleevery 21 days Grade III-IV 9% Nausea/Vomiting 5% Neutropenia Diarrhea 4%Grade III-IV Mucositis 1% 34% Arthralgia/Myalgia 22% Asthenia 14%Peripheral Neuropathy 5% Alopecia 76% Cardiovascular 1% Anemia GradeIII-IV 3% Vinorelbine 30 mg/m² IV weekly Grade III-IV 79% NeutropenicSepsis 3% Nausea/Vomiting (Grade II-IV) 12% Neurologic 9% Diarrhea(Grade II-IV) 4% Alopecia (Grade II-IV) 14% Vinorelbine 30 mg/m² IVweekly Grade III-IV Neutropenic Sepsis 4% Cisplatin 120 mg/m² IV days 1and 29, then 78.7% Nausea/Vomiting every 6 weeks (Grade II-IV) 58%Thrombocytopenia 3% Neurologic 7% Ototoxicity 2% Diarrhea (Grade II-IV)11% Renal (creatinine > 250 umol/L) 6% LUNG CANCER: SMALL-CELLCyclophosphamide 1000 mg/m² IV day 1 Leukopenia Neutropenic InfectionDoxorubicin 45 mg/m² IV day 1 Grade III 33% (Grade III) 9% Etoposide 100mg/m² IV days 1, 3, 5 Grade IV 46% (Grade IV) 3% Repeat cycle every 21days (Grade V) 2% Overall Infection Rate 40% Thrombocytopenia 23%Nausea/Vomiting 33% Mucositis 3% Diarrhea 2% Cardiac 2% Alopecia 57%Cyclophosphamide 800 mg/m² IV day 1 Leukopenia Nausea/VomitingDoxorubicin 50 mg/m² IV day 1 Grade III-IV (Grade II-IV) 64% Vincristine1.4 mg/m² IV day 1 79% Thrombocytopenia 13% Repeat cycle every 21-28days Neuropathy (Grade II-IV) 9% Hepatic (Grade II-V) 11% Renal (GradeII-IV) 4% Alopecia (Grade II-IV) 72% Etoposide 100 mg/m²/d IV days 1-3Leukopenia Neutropenic Infection Carboplatin 300 mg/m² IV day 1 GradeIII 10% (all grades) 11% Repeat cycle every 21 days Grade IV 7%Thrombocytopenia (Grade IV) 4% Alopecia (Grade III) 7% Etoposide 100mg/m²/d IV days 1-3 Neutropenia Documented Infections Cisplatin 100mg/m² IV day 2 Grade III-IV (all grades) 8% Repeat cycle every 28 days85% Fever (all grades) 18% Nausea/Vomiting 19% Thrombocytopenia 18%Creatinine Increase 12% Bilirubin Increase 2% Anemia Grade III-IV 18%Etoposide 100 mg/m²/d IV days 1-3 Neutropenia Documented InfectionsCisplatin 100 mg/m² IV day 2 Grade III-IV (all grades) 22%Cyclophosphamide 400 mg/m²/d IV days 1-3 99% Fever (all grades) 70%Epirubicin 40 mg/m² IV day 1 Nausea/Vomiting 22% Repeat cycle every 28days Thrombocytopenia 78% Creatinine Increase 6% Bilirubin Increase 1%Anemia Grade III-IV 51% Topotecan 1.5 mg/m²/d IV over 30 minutes, GradeIII-IV 89% Neutropenic Fever 28% days 1-5 Neutropenic Sepsis 5% Repeatcycle every 21 days Thrombocytopenia 58% Nausea 4% Fatigue 5% Vomiting2% Stomatitis 2% Anorexia 1% Diarrhea 1% Anemia Grade III-IV 42%Topotecan 1.0 mg/m²/d IV days 1-5 Grade IV 96% Neutropenic FeverPaclitaxel 135 mg/m² IV over 24 hr day 5 Primary (of cycles) 21% Repeatcycle every 28 days prophylaxis Thrombocytopenia with G-CSF (Grade IV)18% Allergic Reaction* 7% Cardiac Dysrhythmia* 4% *Grade not specifiedLYMPHOMA: HODGKIN'S DISEASE Doxorubicin 25 mg/m² IV days 1, 15 GradeIII-IV 21% Neutropenic Infection 2% Bleomycin 10 units/m² IV days 1, 15Thrombocytopenia Vinblastine 6 mg/m² IV days 1, 15 (Grade IV) 5%Dacarbazine 350-375 mg/m² IV days 1, 15 Alopecia 24% Repeat cycle every28 days Pulmonary Toxicity 7% Neuropathy 1% Nausea/Vomiting 33% AnemiaGrade III-IV 5% Chlorambucil 6 mg/m²/d PO days 1-14, LeukopeniaNeutropenic Infection 3% max 10 mg/day Grade III 7% Thrombocytopenia 5%Vinblastine 6 mg/m² IV days 1, 8, Grade IV 2% Nausea/Vomiting 2% max 10mg/day Alopecia <1% Procarbazine 100 mg/m²/d PO days 1-14, max 150mg/day Prednisolone 40 mg/d PO days 1-14 Repeat cycle every 28 daysMechlorethamine 6 mg/m² IV days 1, 8 Grade III-IV 68% NeutropenicInfection Vincristine 1.4 mg/m² IV days 1, 8 (Sepsis) Grade IV 12%(maximum 2 mg) Thrombocytopenia 52% Procarbazine 100 mg/m²/d PO days1-14 Nausea/Vomiting 28% Prednisone 40 mg/m²/d PO days 1-14 PeripheralNeuropathy Repeat cycle every 28 days 8% Alopecia 5% Pulmonary Toxicity4% Anemia Grade III-IV 43% Mechlorethamine 6 mg/m² IV day 1 Grade III37% Neutropenic Fever 17% Doxorubicin 25 mg/m² IV days 1, 15 Grade IV45% Constipation/Ileus* 11% Vinblastine* 6 mg/m² IV days 1, 15 Deep VeinVincristine* 1.4 mg/m² IV days 8, 22 Thrombosis* 3% Bleomycin 5 units/m²IV days 8, 22 Neurologic: Etoposide 60 mg/m² IV days 15, 16 Motor 8%Prednisone 40 mg/m² PO QOD, dose Sensory 11% tapered over the last 15days Autonomic 12% Repeat cycle every 28 days Phlebitis 38% *Inpatients >50 years of age, vinblastine Nausea/Vomiting 9% dose decreasedto 4 mg/m², and vincristine Anemia Grade III-IV dose decreased to 1mg/m² on weeks 9-12. 26% Concomitant trimethoprim/sulfamethoxazole*Required hospitalization DS PO bid; acyclovir 200 mg PO tid,ketoconazole 200 mg PO qd and stool softeners used LYMPHOMA:NON-HODGKIN'S Cyclophosphamide 750 mg/m² IV day 1 Grade IV 91%Doxorubicin 50 mg/m² IV day 1 (Note: Vincristine 1.4 mg/m² IV day 1combined data (maximum 2 mg) w/CNOP) Prednisone 100 mg/d PO days 1-5Repeat cycle every 21 days Rituximab 375 mg/m² IV Neutropenia Sepsis9.1% day 1 of each cycle followed by Grade III 15.1% Fever 9.1%Cyclophosphamide 750 mg/m² IV Grade IV 57.6% Thrombocytopenia day 3 ofcycle Leukopenia (Grade III) 3% Doxorubicin 50 mg/m² IV day 3 of cycleGrade III 9.1% Bowel Obstruction Vincristine 1.4 mg/m² (max 2 mg) IVGrade IV 12.1% (Grade III) 3% day 3 of cycle Intestinal PerforationPrednisone 100 mg/d PO on days 3-7 (Grade IV) 3% of cycleInfusion-Related Repeat cycle every 21 days Reaction Premedicate withacetaminophen and (Grade IV) 3% diphenhydramine prior to rituximabAnemia Grade III 12.1% Cyclophosphamide 750 mg/m² IV day 1 Grade III-IV*Stomatitis 3% Mitoxantrone 10 mg/m² IV day 1 >50% Nausea/Vomiting 16%Vincristine 1.4 mg/m² IV day 1 *Mean ANC Alopecia 24% (maximum 2 mg)600/mm³ Cardiac 2% Prednisone 50 mg/m²/d PO days 1-5 Neurologic 7%Repeat cycle every 21 days Diarrhea 2% Cutaneous 2 Cyclophosphamide 800mg/m² IV day 1, Neutropenia Thrombocytopenia then 200 mg/m²/d IV days2-5 Grade IV (Grade III) Vincristine 1.5 mg/m² IV days 1, 8 in cycle 197.6% cycles 17.1% (<18 years) and and on days 1, 8, and 15 on cycle 3(<18 years) 9.3% (≧18 years) Doxorubicin 40 mg/m² IV day 1 Grade IVThrombocytopenia Cytarabine intrathecal 70 mg days 1, 3 (≧18 years)(Grade IV) Methotrexate 1200 mg/m² IV over 1 hour, 97.8% 53.7% (<18years) and followed by 240 mg/m²/hour for the next Leukopenia 39.5% (≧18years) 23 hours starting on day 10 Grade IV Stomatitis (Grade III)Calcium leucovorin 192 mg/m² IV to start 97.6% cycles 26.8% (<18 years)and 12 hours after the completion of the (<18 years) 28.9% (≧18 years)methotrexate infusion × 1 dose, then Grade IV Stomatitis (Grade IV) 12mg/m² IV every 6 hours until the 95.6% cycles 41.5% (<18 years) andmethotrexate level is <5 × 10−8 mol/L (≧18 years) 20% (≧18 years) GM-CSF7.5 mcg/kg/d subcutaneously Hepatotoxicity (Grade day 13 onwards untilANC >1000. III) Methotrexate 12 mg intrathecal (IT) day 15 24.4% (allages) Hepatotoxicity (Grade IV) 2.4% (all ages) Denileukin diftitox 9 or18 mcg/kg/d IV 9 mcg/kg: Infection 33-43% days 1-5 Leukopenia 6%Infusion-Related Events Repeat cycle every 21 days Neutropenia 3% BackPain 6-11% Premedicate with acetaminophen 18 mcg/kg: Chest Pain/ andantihistamines Leukopenia 3% Tightness 9% Premedication withcorticosteroids Neutropenia 3% Pruritis 6% not permitted Hypotension8-11% Vasodilation 3% Dyspnea 6-11% Constitutional Symptoms 37-47%Gastrointestinal 20-36% Vascular Leak Syndrome 17-25% Thrombotic Events3-8% Rash 19-23% Right Heart Failure 3-6% CNS 17--25% Dexamethasone 40mg/d PO or IV days 1-4 Grade IV 53% Neutropenic Sepsis 31% Cisplatin 100mg/m²/d IV continuous Infection* 31% infusion day 1 Thrombocytopenia*39% Cytarabine 2000 mg/m² IV every 12 hours Renal** 20% for 2 doses, day2 Gastrointestinal† 20% Repeat cycle every 21 to 28 days Neurological††6% Respiratory†† 6% Tumor Lysis Syndrome 5% *Grade IV toxicity**SCr >double baseline value †Severe ††Unknown grade of toxicityEtoposide 40 mg/m²/d IV days 1-4 Grade IV 50% Febrile Neutropenia 30%Methylprednisolone 250-500 mg/d IV Thrombocytopenia days 1-5 (plt<70,000/mm³) 50% Cisplatin 25 mg/m²/d IV continuous infusionNausea/Vomiting 6% days 1-4 Renal (SCr > double Cytarabine 2000 mg/m² IVday 5 baseline) 22% Give immediately following completion of etoposideand cisplatin Repeat cycle every 21-28 days Etoposide 100 mg/m²/d IVdays 1-3 Neutropenia Thrombocytopenia Carboplatin AUC of 5 (maximum dose800 mg) 20.8% 29.4% IV day 2 Neurotoxicity Ifosfamide 5 g/m² IVcontinuous infusion (not graded) 3% over 24 hours day 2 (with 100%replacement Increased Alkaline with mesna) Phosphatase (2x) 11% G-CSF 5mcg/kg/d SQ days 5-12 Increased Alanine Transferase (2x) 9.8% AnemiaGrade III-IV 26.5% Cyclophosphamide 1000 mg/m² IV day 1 NeutropeniaInfectious toxicity 11% Fludarabine 20 mg/m²/d IV (all cycles) Pulmonarytoxicity 18% over 30 minutes days 1-5 Grade III 25% Repeat cycle every28 days of cycles Grade IV 18% of cycles Neutropenia (first cycle,without growth factor) Grade III 26% Grade IV 28% Fludarabine 25 mg/m²/dIV days 1-3 Hematologic Nausea 72.5% Idarubicin 12 mg/m² IV day 1toxicity Repeat cycle every 28 days Grade III-IV 3.7% Fludarabine 20mg/m²/d IV days 1-5 Neutropenia Thrombocytopenia Cyclophosphamide 600mg/m² IV day 1 Grade III 20% (Grade III) 1.6% G-CSF 5 mcg/kg SQ startingday 8 continuing Grade IV 8% Pulmonary 10-14 days until ANC ≧10,000(Grade III) 3.3% Trimethoprim/Sulfamethoxazole PO twice Anemia GradeIII-IV 7% daily every Monday, Wednesday, Friday Allopurinol 300 mg dailydays 1-7, cycle 1 only Repeat cycle every 28 days Fludarabine 25 mg/m²/dIV days 1-3 Neutropenia Febrile Neutropenia 3.8% Mitoxantrone 10 mg/m²IV day 1 Grade IV 20% Thrombocytopenia: Dexamethasone 20 mg/d PO days1-5 Platelets <50,000 8% Repeat cycle every 28 days Platelets <100,00031% Etoposide 60 mg/m²/d IV over 1 hour Neutropenia Thrombocytopeniadays 1-5 Grade IV 100% (Grade III): Ifosfamide 1500 mg/m²/d IV over 1hour (all ages) 14.3% cycles days 1-5 Leukopenia (<18 years) Mesna 360mg/m² IV over 1 hour Grade IV 100% 3.7% (≧18 years) (1 dose mixed withifosfamide), followed by (all ages) Thrombocytopenia mesna 360 mg/m² IVover 15 minutes (Grade IV) every 3 hours days 1-5 82.9% cyclesCytarabine 2000 mg/m² IV over 3 hours every (<18 years) 12 hours on days1, 2 (a total of 4 doses) 96.3% (≧18 years) Methotrexate intrathecal 12mg × 1 on day 5 Hepatotoxicity (Grade GM-CSF 7.5 mcg/kg/d SQ day 7onwards III) 5.9% cycles (<18 years) Stomatitis (Grade III) 5.7% cycles(<18 years) 3.4% cycles (≧18 years) Stomatitis (Grade IV) 2.9% cycles(<18 years) Methotrexate 400 mg/m² IV weeks 2, 6, 10 Grade IV 21%Neutropenic Infection with leucovorin rescue Major Infection 11%Doxorubicin 50 mg/m² IV Minor Infection 11% weeks 1, 3, 5, 7, 9, 11Thrombocytopenia* 2% Cyclophosphamide 350 mg/m² IV Mucositis† 26% weeks1, 3, 5, 7, 9, 11 Neurologic† 8% Vincristine 1.4 mg/m² IV weeks 2, 4, 6,8, Cutaneous† 3% 10, 12 (maximum of 2 mg) Endocrinologic 5% Prednisone75 mg/d PO for 12 weeks then *Grade IV Toxicity taper over last 2 weeks†Severe or Major Bleomycin 10 units/m² IV weeks 4, 8, 12 Anemia GradeIII-IV Trimethoprim/sulfamethoxazole DS tablet PO 20% Requiring bid for12 weeks transfusion Ketoconazole 200 mg/d PO Administer one cycleMethotrexate 200 mg/m² IV days 8, 15 Leukopenia Neutropenic Leucovorin10 mg/m² PO q6h for 6 doses, Grade III 34% Infection* 40% beginning 24hours after each Grade IV 50% Neutropenia methotrexate dose Grade III25% Bleomycin 4 units/m² IV day 1 Grade IV 10% Doxorubicin 45 mg/m² IVday 1 Thrombocytopenia 13% Cyclophosphamide 600 mg/m² IV day 1 Vomiting11% Vincristine 1 mg/m² IV day 1 (maximum 2 mg) Stomatitis 37%Dexamethasone 6 mg/m²/d PO days 1-5 Neurologic 11% Repeat cycle every 21days Pulmonary* 11% Diarrhea 2% Skin 1% Cardiac 3% Hepatic 1% AnemiaGrade III-IV 42% *Grade III-V toxicity Mesna 1330 mg/m²/d IVadministered at Neutropenic Fever same time as ifosfamide, then 500 mgPO, 42% of patients, 4 hours after ifosfamide, days 1-3 7% of cyclesIfosfamide 1330 mg/m²/d IV over 1 hour, Documented Infection days 1-317% of patients, Mitoxantrone 8 mg/m² IV day 1 3% of cycles Etoposide 65mg/m²/d IV days 1-3 Neutrophils* 1.1K Repeat cycle every 21 days for 6cycles, Platelets* 133K followed by 3 to 6 cycles of ESHAP Hemoglobin*9.7 g/dl Nausea/Vomiting 11% Diarrhea 3% Stomatitis 1% Hearing Loss 2%Cardiovascular 2% *Mean nadir levels Prednisone 60 mg/m²/d PO day 1-14Grade III-IV 20% Febrile Neutropenia 20% Doxorubicin 25 mg/m² IV day 1Documented Cyclophosphamide 650 mg/m² IV day 1 Infection 13% Etoposide120 mg/m² IV day 1 Thrombocytopenia 7% Cytarabine 300 mg/m² IV day 8Tumor Lysis 16% Bleomycin 5 units/m² IV day 8 Coagulopathy* 12%Vincristine 1.4 mg/m² IV day 8 GI Bleeding* 5% Methotrexate 120 mg/m² IVday 8 Anemia Grade III-IV Leucovorin 25 mg/m² PO q6h for 4 doses 61%Requiring beginning 24 hours after methotrexate transfusionTrimethoprim/sulfamethoxazole DS PO bid *Requiring Repeat cycle every 21to 28 days hospitalization MELANOMA Cisplatin 20 mg/m²/d IV days 2-5Grade IV 50% Neutropenic Fever 30% Vinblastine 1.6 mg/m²/d IV days 1-5Thrombocytopenia 26% Dacarbazine 800 mg/m² IV day 1 Alopecia* 100%Repeat cycle every 21 days Nausea/Vomiting* 86% Renal* 12% Diarrhea* 34%Hypomagnesemia** 68% Neuropathy† 20% *Unknown grade **Requiringreplacement †Moderately severe Dacarbazine 1000 mg/m² IV day 1Neutropenia Thrombocytopenia 7% Repeat cycle every 21 days Grade III 10%Nausea/Vomiting 5% Grade IV 9% Dyspnea 1% Leukopenia Anemia Grade III-IV6% without neutropenia Grade III 1% Dacarbazine 250 mg/m²/d IVNeutropenia Fever (Grade III) 2% over 30 minutes days 1-5 Grade III 1%Thrombocytopenia 8% Repeat cycle every 21 days Grade IV 1% Asthenia(Grade III) 1% Fatigue (Grade III) 2% Headache (Grade III) 1% Pain(Grade III) 13% Constipation (Grade III) 3% Nausea (Grade III) 4%Vomiting (Grade III) 4% Somnolence (Grade III) 1% Anemia Grade IV 1%Dacarbazine 220 mg/m²/d IV days 1-3, Grade IV Neutropenic Fever 11%every 22-24 days 31%-32% Systemic Infection Carmustine 150 mg/m² IV day1 (requiring IV Cisplatin 25 mg/m²/d IV days 1-3, antibiotics) 4% or 3%every 22-24 days Deep Vein Thrombosis Tamoxifen 160 mg/d PO 7 daysbefore 4% chemotherapy, then 40 mg/d days 1-42 Hot Flashes 3% Repeatevery 43 days Thrombocytopenia* 44% Vomiting† 40% *Grade IV toxicity†Unknown toxicity grade Tamoxifen 10 mg PO twice daily NeutropeniaThrombocytopenia 57% (start 1 week prior to chemotherapy and Grade III24% Nausea/Vomiting 18% continue indefinitely) Grade IV 15% Fatigue 7%Carmustine (BCNU) 150 mg/m² IV day 1 Leukopenia Increased Serum (repeatevery 42 days-every other cycle) without Creatinine 3% Cisplatin 25mg/m²/d IV days 1-3 neutropenia Dyspnea 5% Dacarbazine 220 mg/m²/d IVdays 1-3 Grade III 8% Anemia Grade III-IV Repeat cisplatin anddacarbazine Grade IV 1% 32% every 21 days Dacarbazine 800 mg/m² IV day 1Neutropenia Neutropenic Fever 9% Cisplatin 20 mg/m²/d IV days 1-4 GradeIV 30% Infection 7% Vinblastine 1.2 mg/m²/d IV days 1-4 Thrombocytopenia43% Interferon-alfa 2b 5 MU/m²/d SQ Hypotension 30% days 1-5, 8, 10, 12Nausea/Vomiting 27% Aldesleukin 9 MU/m²/d IV continuous RenalInsufficiency 11% infusion days 1-4 Neurologic Toxicity 5% Filgrastim 5mcg/kg/d SQ days 7-16 Bleeding 2% Repeat cycle every 21 days for amaximum of 4 cycles Temozolomide 200 mg/m²/d PO days 1-5 NeutropeniaFever 2% Repeat cycle every 28 days Grade III 1% Thrombocytopenia 7%Grade IV 2% Asthenia (Grade III) 3% Fatigue (Grade III) 3% Headache 6%Pain (Grade III) 7% Constipation (Grade III) 3% Nausea (Grade III) 4%Vomiting 5% Anemia Grade III-IV 2% MULTIPLE MYELOMA Vincristine 0.03mg/kg IV day 1 Leukopenia Parethesias and Carmustine 0.5-1 mg/kg IV day1 Grade IV 16% peripheral neuropathy Cyclophosphamide 10 mg/kg IV day 1occurred frequently Melphalan 0.25 mg/kg/d PO days 1-4 or Nauseaoccurred 0.1 mg/kg/d PO days 1-7 or 1-10 occasionally Prednisone 1mg/kg/d PO days 1-7 Repeat cycle every 35-42 days Melphalan 8 mg/m²/d POdays 1-4 Grade III 29% Neutropenic Infections Prednisone 60 mg/m²/d POdays 1-4 Grade IV 8% (Grade III-IV) 14% Repeat cycle every 28 daysNausea/Vomiting* 10% Neuropathy* 2% Alopecia† 7% Thrombocytopenia 23%*Grade II-IV toxicity †Grade I-IV toxicity Thalidomide 200 mg PO dailyat bedtime Thrombocytopenia 3.6% (dose increase every 2 weeks for 6weeks Anemia Grade III-IV up to a dose of 800 mg/day) 3.6% Vincristine0.4 mg/d IV continuous infusion Neutropenic Sepsis 30% days 1-4Thrombocytopenia Doxorubicin 9 mg/m²/d IV continuous (Grade II) 11%infusion days 1-4 Removal of Catheter Dexamethasone 40 mg PO Due toThrombis qd 1-4, 9-12, 17-20 or Infection 14% Repeat cycle every 28-35days Cushingoid Features 26% Irritability 19% Aggravation of Diabetes11% Vincristine 1.2 mg/m² IV Grade III 30% Neutropenia day 1 (maximum 2mg) Grade IV 16% (Grade IV) 14% Carmustine 20 mg/m² IV day 1Nausea/Vomiting* 31% Melphalan 8 mg/m²/d PO days 1-4 Neuropathy* 24%Cyclophosphamide 400 mg/m² IV day 1 Alopecia† 25% Prednisone 40 mg/m²/dPO Thrombocytopenia 23% days 1-7 of all cycles *Grade II-IV toxicity 20mg/m²/d PO days 8-14 †Grade I-IV toxicity first three cycles only Repeatcycle every 35 days SARCOMA: UNCLASSIFIED Doxorubicin 22.5 mg/m²/d IVcontinuous Grade III-IV 38% Nausea/Vomiting 9% infusion days 1-4Thrombocytopenia 4% Dacarbazine 225 mg/m²/d IV continuous Cardiac 1%infusion days 1-4 Pulmonary Embolism 1% Repeat cycle every 21 daysMucositis/Stomatitis 7% Anemia Grade III-IV 6% Doxorubicin 50 mg/m² IVbolus, day 1 Leukopenia Neutropenic Fever 2% Ifosfamide 5000 mg/m²/d IVcontinuous Grade III-IV Documented Infection infusion followingdoxorubicin, day 1 73% 6% Mesna 600 mg/m² IV bolus before Grade IV 34%Fever 2% ifosfamide, 2500 mg/m²/d IV continuous Thrombocytopenia* 5%infusion with ifosfamide 1250 mg/m² IV Nausea/Vomiting 41% over 12 hrfollowing ifosfamide Alopecia 71% Repeat cycle every 21 days Diarrhea 2%Oral 3% Renal 2% Cardiac 1% Consciousness 3% Hematuria 2% *<75,000/mm³Doxorubicin 75 mg/m² IV day 1 Grade III-IV 51% Infection 3% Repeat cycleevery 21 days Grade IV 21% Nausea/Vomiting 13% Thrombocytopenia 2%Mucositis 6% Cardiotoxicity 1% Etoposide 100 mg/m²/d IV days 1-3Leukopenia Neutropenic Fever Ifosfamide 2500 mg/m²/d IV days 1-3 GradeIV 38% during Leukopenia 5% Mesna at 20% of ifosfamide doseNausea/Vomiting prior to and 4, 8, and 12 hours after (all Grades) 70%ifosfamide administration Thrombocytopenia 3% Repeat cycle every 28 daysAlopecia (significant) 85% Renal 2% Mesna 2500 mg/m²/d IV continuousGrade III-IV 80% Neutropenic Infection 10% infusion days 1-4Thrombocytopenia 26% Doxorubicin 15 mg/m²/d IV continuousSomnolence/Coma 1% infusion days 1-4 Nausea/Vomiting 9% Ifosfamide 2500mg/m²/d IV continuous Mucositis/Stomatitis 7% infusion days 1-3Gastrointestinal 4% Dacarbazine 250 mg/m²/d IV continuous Anemia GradeIII-IV infusion days 1-4 22% Repeat cycle every 21 days SARCOMA:AIDS-RELATED KAPOSI'S SARCOMA Liposomal daunorubicin 40 mg/m² IVLeukopenia Fever 5% over 30-60 minutes every 2 weeks Grade III 33%Thrombocytopenia 1% Grade IV 5% Fatigue 6% Neutropenia Diarrhea 4% GradeIII 36% Nausea/Vomiting 5% Grade IV 15% Abdominal Pain 3% Dyspnea 3%Allergic Reactions 3% Anemia Grade III-IV 11% Liposomal doxorubicin 20mg/m² IV Leukopenia 36% Thrombocytopenia 3% over 30 minutes every 2weeks Neutropenia Nausea/Vomiting 15% Grade IV 6% Peripheral Neuropathy6% Mucositis 5% Anemia Grade III-IV 9.8% Paclitaxel 100 mg/m² IVNeutropenia Thrombocytopenia 6% over 3 hours every 2 weeks Grade III 25%Increased AST 5% Grade IV 36% Alopecia (Grade III) 9% Fatigue 25%Myalgia (Grade III) 16% Nausea/Vomiting (Grade III) 13% Diarrhea 16%Neuropathy (Grade III) 2% Mucositis (Grade III) 2% Anemia Grade III-IV27% UNKNOWN PRIMARY: ADENOCARCINOMA 5-Fluorouracil 600 mg/m² IV days 1,8, 29, 36 Leukopenia Neutropenic Sepsis 5% Doxorubicin 30 mg/m² IV days1, 29 Grade III-IV 7% Thrombocytopenia 14% Mitomycin 10 mg/m² IV day 1Mucositis (Grade II-III) Repeat cycle every 8 weeks 7% Cardiac 2%Paclitaxel 200 mg/m² IV Leukopenia Neutropenic Fever 13% over 1 hour,day 1, followed by Grade III 56% Nausea/Vomiting 9% carboplatin dosetargeted by Grade IV 20% Thrombocytopenia 26% Calvert equation to AUC of6 IV day 1 Peripheral Neuropathy Etoposide 50 mg/d PO alternated with 7%100 mg/d PO days 1-10 Hypersensitivity 2% Repeat cycle every 21 days

In certain embodiments, patients are subjected to a hypoxia imagingtechnique prior to administration of the compositions comprising thecompounds of the present invention. Examples of imaging techniquessuitable for the determination of the presence of hypoxic tumor cellsinclude computed tomography (CT), magnetic resonance imaging (MRI),single photon emission computer tomography (SPECT), and positronemission tomography (PET). Use of such visualization methods canadvantageously be used to select a subset of patients that areparticularly suitable for treatment with hypoxia activatedantiproliferative compositions of the present invention.

In this embodiment, the invention is directed to a method of treating,preventing or ameliorating a hyperproliferative disease in an animal inneed thereof, comprising determining whether said hyperproliferativedisease is characterized by hypoxic tissue, and treating said animalwith an effective amount of a compound of the invention.

The term “radiotherapeutic agent,” as used herein, is intended to referto any radiotherapeutic agent known to one of skill in the art to beeffective to treat or ameliorate cancer, without limitation. Forinstance, the radiotherapeutic agent can be an agent such as thoseadministered in brachytherapy or radionuclide therapy. Such methods canoptionally further comprise the administration of one or more additionalcancer therapies, such as, but not limited to, chemotherapies, surgery,and/or another radiotherapy.

In certain embodiments involving radiotherapeutic agents or treatments,the present invention relates to a method for treating cancer comprisingthe administration of vinca alkaloid or analog N-oxide having Formula I,in combination with a treatment comprising a therapeutically effectivedose of brachytherapy. The brachytherapy can be administered accordingto any schedule, dose, or method known to one of skill in the art to beeffective in the treatment or amelioration of cancer, withoutlimitation. In general, brachytherapy comprises insertion of radioactivesources into the body of a subject to be treated for cancer, preferablyinside the tumor itself, such that the tumor is maximally exposed to theradioactive source, while preferably minimizing the exposure of healthytissue.

In certain embodiments, the brachytherapy can be intracavitarybrachytherapy. In other embodiments, the brachytherapy can beinterstitial brachytherapy. Whether the brachytherapy is intracavitarybrachytherapy or interstitial brachytherapy, the brachytherapy can beadministered at a high dose rate, a continuous low dose rate, or apulsed dose rate. For example, and not by way of limitation, a high doserate brachytherapy regimen can be a dose of 60 Gy administered in tenfractions over six days, while a continuous low dose rate brachytherapyregimen can be a total dose of about 65 Gy, administered continuously atabout 40 to 50 cGy per hour. Other examples of high, continuous low, andpulsed dose rate brachytherapy are well known in the art. See, e.g.,Mazeron et al., Sem. Rad. One. 12:95-108 (2002).

Representative radioisotopes that can be administered in any of theabove-described brachytherapies include, but are not limited to,phosphorus 32, cobalt 60, palladium 103, ruthenium 106, iodine 125,cesium 137, iridium 192, xenon 133, radium 226, californium 252, or gold198. Other radioisotopes may be selected for administration inbrachytherapy according to the desirable physical properties of such aradioisotope. One of skill in the art will readily recognize that manyproperties will affect a radioisotope's suitability for use inbrachytherapy, including, but not limited to, the radioisotope'shalf-life, the degree to which emitted radiation penetrates surroundingtissue, the energy of emitted radiation, the ease or difficulty ofadequately shielding the radioisotope, the availability of theradioisotope, and the ease or difficulty of altering the shape of theradioisotope prior to administration.

Additional methods of administering and apparatuses and compositionsuseful for brachytherapy are described in U.S. Pat. Nos. 6,319,189,6,179,766, 6,168,777, 6,149,889, and 5,611,767, each of which isincorporated herein by reference in its entirety.

In certain embodiments involving radiotherapeutic agents or treatments,the present invention relates to a method for treating cancer comprisingthe administration of vinca alkaloid or analog N-oxide having Formula I,in combination with a treatment comprising a therapeutically effectivedose of a radionuclide. The radionuclide therapy can be administeredaccording to any schedule, dose, or method known to one of skill in theart to be effective in the treatment or amelioration of cancer, withoutlimitation. In general, radionuclide therapy comprises systemicadministration of a radioisotope that preferentially accumulates in orbinds to the surface of cancerous cells. The preferential accumulationof the radionuclide can be mediated by a number of mechanisms,including, but not limited to, incorporation of the radionuclide intorapidly proliferating cells, specific accumulation of the radionuclideby the cancerous tissue without special targeting (e.g., iodine 131accumulation in thyroid cancer), or conjugation of the radionuclide to abiomolecule specific for a neoplasm.

Representative radioisotopes that can be administered in radionuclidetherapy include, but are not limited to, phosphorus 32, yttrium 90,dysprosium 165, indium 111, strontium 89, samarium 153, rhenium 186,iodine 131, iodine 125, lutetium 177, and bismuth 213. While all ofthese radioisotopes may be linked to a biomolecule providing specificityof targeting, iodine 131, indium 111, phosphorus 32, samarium 153, andrhenium 186 may be administered systemically without such conjugation.One of skill in the art may select a specific biomolecule for use intargeting a particular neoplasm for radionuclide therapy based upon thecell-surface molecules present on that neoplasm. For example, hepatomasmay be specifically targeted by an antibody specific for ferritin, whichis frequently over-expressed in such tumors. Examples ofantibody-targeted radioisotopes for the treatment of cancer includeZEVALIN (ibritumomab tiuxetan) and BEXXAR (tositumomab), both of whichcomprise an antibody specific for the B cell antigen CD20 and are usedfor the treatment of non-Hodgkin lymphoma.

Other examples of biomolecules providing specificity for particular cellare reviewed in an article by Thomas, Cancer Biother. Radiopharm.17:71-82 (2002), which is incorporated herein by reference in itsentirety. Furthermore, methods of administering and compositions usefulfor radionuclide therapy may be found in U.S. Pat. Nos. 6,426,400,6,358,194, 5,766,571, and 5,563,250, each of which is incorporatedherein by reference in its entirety.

In certain embodiments involving radiotherapeutic agents or treatments,the present invention relates to a method for treating cancer comprisingthe administration of vinca alkaloid or analog N-oxide having Formula I,in combination with a treatment comprising a therapeutically effectivedose of external-beam radiation therapy. The external-beam radiationtherapy can be administered according to any schedule, dose, or methodknown to one of skill in the art to be effective in the treatment oramelioration of cancer, without limitation. In general, external-beamradiation therapy comprises irradiating a defined volume within asubject with a high energy beam, thereby causing cell death within thatvolume. The irradiated volume preferably contains the entire cancer tobe treated, and preferably contains as little healthy tissue aspossible.

In certain embodiments, the external-beam radiation therapy can bethree-dimensional conformal radiotherapy. In other embodiments, theexternal-beam radiation therapy can be continuous hyperfractionatedradiotherapy. In still other embodiments, the external-beam radiationtherapy can be intensity-modulated radiotherapy. In yet otherembodiments, the external-beam radiation therapy can be helicaltomotherapy. In still other embodiments, the external-beam radiationtherapy can be three-dimensional conformal radiotherapy with doseescalation. In yet other embodiments, the external-beam radiationtherapy can be stereotactic radiotherapy, including, but not limited to,single fraction stereotactic radiotherapy, fractionated stereotacticradiotherapy, and fractionated stereotactically guided conformalradiotherapy.

The external-beam radiation therapy can be generated or manipulated byany means known to one of skill in the art. For example, the photon beamused in external-beam radiation therapy can be shaped by a multileafcollimator. Other examples of suitable devices for generating a photonbeam for use in external-beam radiation therapy include a gamma knifeand a linac-based stereotactic apparatus. In certain embodiments,administration of the external-beam radiation therapy is controlled by acomputer according to a three-dimensional model of the patient in thetreatment position. Such a model can be generated, for example, bycomputed tomography (CT), magnetic resonance imaging (MRI), singlephoton emission computer tomography (SPECT), and positron emissiontomography (PET). Use of such visualization methods can advantageouslyminimize the volume of healthy tissue treated, thereby allowing highertotal doses of radiation to be administered to the patient.

In addition, healthy tissues can optionally be protected from theeffects of the external-beam radiation therapy by placing blockingdevices such as, e.g., lead shields, in locations where such protectionis needed. Alternatively or additionally, metal reflecting shields canoptionally be located to reflect the photon beam in order to concentratethe radiation on the cancerous tissue to be treated and protect healthytissue. Placement of either shield is well within the knowledge of oneof skill in the art.

Methods of administering and apparatuses and compositions useful forexternal-beam radiation therapy can be found in U.S. Pat. Nos.6,449,336, 6,398,710, 6,393,096, 6,335,961, 6,307,914, 6,256,591,6,245,005, 6,038,283, 6,001,054, 5,802,136, 5,596,619, and 5,528,652,each of which is incorporated herein by reference in its entirety.

In certain embodiments involving radiotherapeutic agents or treatments,the present invention relates to a method for treating cancer comprisingthe administration of vinca alkaloid or analog N-oxide having Formula I,in combination with a treatment comprising a therapeutically effectivedose of thermotherapy. The thermotherapy can be administered accordingto any schedule, dose, or method known to one of skill in the art to beeffective in the treatment or amelioration of cancer, withoutlimitation. In certain embodiments, the thermotherapy can becryoablation therapy. In other embodiments, the thermotherapy can behyperthermic therapy. In still other embodiments, the thermotherapy canbe a therapy that elevates the temperature of the tumor higher than inhyperthermic therapy.

Cryoablation therapy involves freezing of a neoplastic mass, leading todeposition of intra- and extra-cellular ice crystals; disruption ofcellular membranes, proteins, and organelles; and induction of ahyperosmotic environment, thereby causing cell death. Cryoablation canbe performed in one, two, or more freeze-thaw cycles, and further theperiods of freezing and thawing can be adjusted for maximum tumor celldeath by one of skill in the art. One exemplary device that can be usedin cryoablation is a cryoprobe incorporating vacuum-insulated liquidnitrogen. See, e.g., Murphy et al., Sem. Urol. Oncol. 19:133-140 (2001).However, any device that can achieve a local temperature of about −180°C. to about −195° C. can be used in cryoablation therapy. Methods forand apparatuses useful in cryoablation therapy are described in U.S.Pat. Nos. 6,383,181, 6,383,180, 5,993,444, 5,654,279, 5,437,673, and5,147,355, each of which is incorporated herein by reference in itsentirety.

Hyperthermic therapy typically involves elevating the temperature of aneoplastic mass to a range from about 42° C. to about 44° C. Thetemperature of the cancer may be further elevated above this range;however, such temperatures can increase injury to surrounding healthytissue while not causing increased cell death within the tumor to betreated. The tumor may be heated in hyperthermic therapy by any meansknown to one of skill in the art without limitation. For example, andnot by way of limitation, the tumor may be heated by microwaves, highintensity focused ultrasound, ferromagnetic thermoseeds, localizedcurrent fields, infrared radiation, wet or dry radiofrequency ablation,laser photocoagulation, laser interstitial thermic therapy, andelectrocautery. Microwaves and radiowaves can be generated by waveguideapplicators, horn, spiral, current sheet, and compact applicators.

Other methods of and apparatuses and compositions for raising thetemperature of a tumor are reviewed in an article by Wust et al., LancetOncol. 3:487-97 (2002), and described in U.S. Pat. Nos. 6,470,217,6,379,347, 6,165,440, 6,163,726, 6,099,554, 6,009,351, 5,776,175,5,707,401, 5,658,234, 5,620,479, 5,549,639, and 5,523,058, each of whichis incorporated herein by reference in its entirety.

In certain embodiments involving radiotherapeutic agents or treatments,the present invention relates to a method for treating cancer comprisingthe administration of vinca alkaloid or analog N-oxide having Formula I,in combination with a treatment comprising a therapeutically effectivedose of radiosurgery. The radiosurgery can be administered according toany schedule, dose, or method known to one of skill in the art to beeffective in the treatment or amelioration of cancer, withoutlimitation. In general, radiosurgery comprises exposing a defined volumewithin a subject to a manually directed radioactive source, therebycausing cell death within that volume. The irradiated volume preferablycontains the entire cancer to be treated, and preferably contains aslittle healthy tissue as possible. Typically, the tissue to be treatedis first exposed using conventional surgical techniques, then theradioactive source is manually directed to that area by a surgeon.Alternatively, the radioactive source can be placed near the tissue tobe irradiated using, for example, a laparoscope. Methods and apparatusesuseful for radiosurgery are further described in Valentini et al., Eur.J. Surg. Oncol. 28:180-185 (2002) and in U.S. Pat. Nos. 6,421,416,6,248,056, and 5,547,454, each of which is incorporated herein byreference in its entirety.

In certain embodiments involving radiotherapeutic agents or treatments,the present invention relates to a method for treating cancer comprisingthe administration of vinca alkaloid or analog N-oxide having Formula I,in combination with a treatment comprising a therapeutically effectivedose of charged-particle radiotherapy. The charged-particle radiotherapycan be administered according to any schedule, dose, or method known toone of skill in the art to be effective in the treatment or ameliorationof cancer, without limitation. In certain embodiments, thecharged-particle radiotherapy can be proton beam radiotherapy. In otherembodiments, the charged-particle radiotherapy can be helium ionradiotherapy. In general, charged-particle radiotherapy comprisesirradiating a defined volume within a subject with a charged-particlebeam, thereby causing cellular death within that volume. The irradiatedvolume preferably contains the entire cancer to be treated, andpreferably contains as little healthy tissue as possible. A method foradministering charged-particle radiotherapy is described in U.S. Pat.No. 5,668,371, which is incorporated herein by reference in itsentirety.

In certain embodiments involving radiotherapeutic agents or treatments,the present invention relates to a method for treating cancer comprisingthe administration of vinca alkaloid or analog N-oxide having Formula I,in combination with a treatment comprising a therapeutically effectivedose of neutron radiotherapy. The neutron radiotherapy can beadministered according to any schedule, dose, or method known to one ofskill in the art to be effective in the treatment or amelioration ofcancer, without limitation.

In certain embodiments, the neutron radiotherapy can be a neutroncapture therapy. In such embodiments, a compound that emits radiationwhen bombarded with neutrons and preferentially accumulates in aneoplastic mass is administered to a subject. Subsequently, the tumor isirradiated with a low energy neutron beam, activating the compound andcausing it to emit decay products that kill the cancerous cells. Suchcompounds are typically boron containing compounds, but any compoundthat has a significantly larger neutron capture cross-section thancommon body constituents can be used. The neutrons administered in suchtherapies are typically relatively low energy neutrons having energiesat or below about 0.5 eV. The compound to be activated can be caused topreferentially accumulate in the target tissue according to any of themethods useful for targeting of radionuclides, as described below, or inthe methods described in Laramore, Semin. Oncol. 24:672-685 (1997) andin U.S. Pat. Nos. 6,400,796, 5,877,165, 5,872,107, and 5,653,957, eachof which is incorporated herein by reference in its entirety.

In other embodiments, the neutron radiotherapy can be a fast neutronradiotherapy. In general, fast neutron radiotherapy comprisesirradiating a defined volume within a subject with a neutron beam,thereby causing cellular death within that volume. The irradiated volumepreferably contains the entire cancer to be treated, and preferablycontains as little healthy tissue as possible. Generally, high energyneutrons are administered in such therapies, with energies in the rangeof about 10 to about 100 million eV. Optionally, fast neutronradiotherapy can be combined with charged-particle radiotherapy in theadministration of mixed proton-neutron radiotherapy.

In certain embodiments involving radiotherapeutic agents or treatments,the present invention relates to a method for treating cancer comprisingthe administration of vinca alkaloid or analog N-oxide having Formula I,in combination with a treatment comprising a therapeutically effectivedose of photodynamic therapy. The photodynamic therapy can beadministered according to any schedule, dose, or method known to one ofskill in the art to be effective in the treatment or amelioration ofcancer, without limitation. In general, photodynamic therapy comprisesadministering a photosensitizing agent that preferentially accumulatesin a neoplastic mass and sensitizes the neoplasm to light, then exposingthe tumor to light of an appropriate wavelength. Upon such exposure, thephotosensitizing agent catalyzes the production of a cytotoxic agent,such as, e.g., singlet oxygen, which kills the cancerous cells.

Representative photosensitizing agents that may be used in photodynamictherapy include, but are not limited to, porphyrins such as porfimersodium, 5-aminolaevulanic acid and verteporfin; chlorins such astemoporfin; texaphyrins such as lutetium texephyrin; purpurins such astin etiopurpurin; phthalocyanines; and titanium dioxide. The wavelengthof light used to activate the photosensitizing agent can be selectedaccording to several factors, including the depth of the tumor beneaththe skin and the absorption spectrum of the photosensitizing agentadministered. The period of light exposure may also vary according tothe efficiency of the absorption of light by the photosensitizing agentand the efficiency of the transfer of energy to the cytotoxic agent.Such determinations are well within the ordinary skill of one in theart.

Methods of administering and apparatuses and compositions useful forphotodynamic therapy are disclosed in Hopper, Lancet Oncol. 1:212-219(2000) and U.S. Pat. Nos. 6,283,957, 6,071,908, 6,011,563, 5,855,595,5,716,595, and 5,707,401, each of which is incorporated herein byreference in its entirety.

It will be appreciated that both the particular radiation dose to beutilized in treating a hyperproliferative disorder and the method ofadministration will depend on a variety of factors. Thus, the dosages ofradiation that can be used according to the methods of the presentinvention are determined by the particular requirements of eachsituation. The dosage will depend on such factors as the size of thetumor, the location of the tumor, the age and sex of the patient, thefrequency of the dosage, the presence of other tumors, possiblemetastases and the like. Those skilled in the art of radiotherapy canreadily ascertain the dosage and the method of administration for anyparticular tumor by reference to Hall, E. J., Radiobiology for theRadiologist, 5th edition, Lippincott Williams & Wilkins Publishers,Philadelphia, Pa., 2000; Gunderson, L. L. and Tepper J. E., eds.,Clinical Radiation Oncology, Churchill Livingstone, London, England,2000; and Grosch, D. S., Biological Effects of Radiation, 2nd edition,Academic Press, San Francisco, Calif., 1980. In certain embodiments,radiotherapeutic agents and treatments may be administered at doseslower than those known in the art due to the additive or synergisticeffect of the compound having Formula I.

Compositions in accordance with the present invention may be employedfor administration in any appropriate manner, e.g., oral or buccaladministration, e.g., in unit dosage form, for example in the form of atablet, in a solution, in hard or soft encapsulated form includinggelatin encapsulated form, sachet, or lozenge. Compositions may also beadministered parenterally or topically, e.g., for application to theskin, for example in the form of a cream, paste, lotion, gel, ointment,poultice, cataplasm, plaster, dermal patch or the like, or forophthalmic application, for example in the form of an eye-drop, -lotionor -gel formulation. Readily flowable forms, for example solutions,emulsions and suspensions, may also be employed e.g., for intralesionalinjection, or may be administered rectally, e.g., as an enema orsuppository, or intranasal administration, e.g., as a nasal spray oraerosol. Microcrystalline powders may be formulated for inhalation,e.g., delivery to the nose, sinus, throat or lungs. Transdermalcompositions/devices and pessaries may also be employed for delivery ofthe compounds of the invention. The compositions may additionallycontain agents that enhance the delivery of the compounds having FormulaI (or other active agents), e.g., liposomes, polymers or co-polymers(e.g., branched chain polymers). Preferred dosage forms of the presentinvention include oral dosage forms and intravenous dosage forms.

When the unit dosage of the vinca alkaloid N-oxide, an analog, or a saltthereof is a solution or a suspension, the dosage form may be in asealed container with substantially no carbon dioxide.

Intravenous forms include, but are not limited to, bolus and dripinjections. In preferred embodiments, the intravenous dosage forms aresterile or capable of being sterilized prior to administration to asubject since they typically bypass the subject's natural defensesagainst contaminants. Examples of intravenous dosage forms include, butare not limited to, Water for Injection USP; aqueous vehicles including,but not limited to, Sodium Chloride Injection, Ringer's Injection,Dextrose Injection, Dextrose and Sodium Chloride Injection, and LactatedRinger's Injection; water-miscible vehicles including, but not limitedto, ethyl alcohol, polyethylene glycol and polypropylene glycol; andnon-aqueous vehicles including, but not limited to, corn oil, cottonseedoil, peanut oil, sesame oil, ethyl oleate, isopropyl myristate andbenzyl benzoate. In certain embodiments, vincristine N-oxide forms aclear solution at 5 mg/mL and at about 20 mg/mL in de-ionized water, insaline solution, and in 5% dextrose/water solution.

The pharmaceutical compositions of the present invention may furthercomprise one or more additives. Additives that are well known in the artinclude, e.g., detackifiers, anti-foaming agents, buffering agents,antioxidants (e.g., ascorbic acid, ascorbyl palmitate, sodium ascorbate,butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), propylgallate, malic acid, fumaric acid, potassium metabisulfite, sodiumbisulfite, sodium metabisulfite, and tocopherols, e.g., α-tocopherol(vitamin E)), preservatives, chelating agents, viscomodulators,tonicifiers, flavorants, colorants, odorants, opacifiers, suspendingagents, binders, fillers, plasticizers, lubricants, and mixturesthereof. The amounts of such additives can be readily determined by oneskilled in the art, according to the particular properties desired, andcan be formulated such that compounds having Formula I are stable, e.g.,not reduced by antioxidant additives.

The additive may also comprise a thickening agent. Suitable thickeningagents may be of those known and employed in the art, including, e.g.,pharmaceutically acceptable polymeric materials and inorganic thickeningagents. Exemplary thickening agents for use in the presentpharmaceutical compositions include polyacrylate and polyacrylateco-polymer resins, for example poly-acrylic acid and poly-acrylicacid/methacrylic acid resins; celluloses and cellulose derivativesincluding: alkyl celluloses, e.g., methyl-, ethyl- andpropyl-celluloses; hydroxyalkyl-celluloses, e.g.,hydroxypropyl-celluloses and hydroxypropylalkyl-celluloses such ashydroxypropyl-methyl-celluloses; acylated celluloses, e.g.,cellulose-acetates, cellulose-acetatephthallates,cellulose-acetatesuccinates and hydroxypropylmethyl-cellulosephthallates; and salts thereof such as sodium-carboxymethyl-celluloses;polyvinylpyrrolidones, including for example poly-N-vinylpyrrolidonesand vinylpyrrolidone co-polymers such as vinylpyrrolidone-vinylacetateco-polymers; polyvinyl resins, e.g., including polyvinylacetates andalcohols, as well as other polymeric materials including gum traganth,gum arabicum, alginates, e.g., alginic acid, and salts thereof, e.g.,sodium alginates; and inorganic thickening agents such as atapulgite,bentonite and silicates including hydrophilic silicon dioxide products,e.g., alkylated (for example methylated) silica gels, in particularcolloidal silicon dioxide products.

Such thickening agents as described above may be included, e.g., toprovide a sustained release effect. However, where oral administrationis intended, the use of thickening agents may not be required. Use ofthickening agents is, on the other hand, indicated, e.g., where topicalapplication is foreseen.

In one embodiment of the invention, compounds having Formula I areformulated as described, for example, in U.S. Pat. No. 4,923,876.

Although the dosage of the compound having Formula I will vary accordingto the activity and/or toxicity of the particular compound, thecondition being treated, and the physical form of the pharmaceuticalcomposition being employed for administration, it may be stated by wayof guidance that a dosage selected in the range from 0.1 to 20 mg/kg ofbody weight per day will often be suitable, although higher dosages,such as 0.1 to 50 mg/kg of body weight per day may be useful. Those ofordinary skill in the art are familiar with methods for determining theappropriate dosage. Methods for assessing the toxicity, activity and/orselectivity of the compounds having Formula I may be carried out usingany of the methods known in the art, including the antiproliferativeactivity test.

In certain instances, the dosage of the compounds having Formula I willbe lower, e.g., when used in combination with at least a secondhyperproliferative disorder treatment, and may vary according to theactivity and/or toxicity of the particular compound, the condition beingtreated, and the physical form of the pharmaceutical composition beingemployed for administration.

When the composition of the present invention is formulated in unitdosage form, the compound having Formula I will preferably be present inan amount of between 0.01 and 2000 mg per unit dose. More preferably,the amount of compound having Formula I per unit dose will be about0.01, 0.05, 0.1, 0.5, 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100,150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800,850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400,1450, 1500, 1550, 1600, 1650, 1700, 1750, 1800, 1850, 1900, 1950, 2000,2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3250, 3500,3750, 4000, 4250, 4500, 4750, 5000, 5250, 5500, 5750, 6000, 6250, 6500,6750, 7000, 7250, 7500, 7750, 8000, 8250, 8500, 8750, 9000, 9250, 9500,9750, or 10000 mg or any amount therein.

When the unit dosage form of the composition is a capsule, the totalquantity of ingredients present in the capsule is preferably about10-1000 μL. More preferably, the total quantity of ingredients presentin the capsule is about 100-300 μL. In another embodiment, the totalquantity of ingredients present in the capsule is preferably about10-1500 mg, preferably about 100-1000 mg.

Irinotecan is preferably administered at a dose of about 5 mg/m² toabout 500 mg/m², from about 50 mg/m² to about 300 mg/m², from about 75mg/m² to about 200 mg/m². In a specific embodiment, an effective amountof irinotecan is 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75,80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150,155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220,225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290,295, or 300 mg/m² or more. In certain embodiments, an effective dose ofirinotecan is between about 20 mg to about 500 mg, between about 40 mgto about 300 mg, between about 50 mg to about 200 mg, between about 75mg to about 150 mg. In certain embodiments, the methods of the inventioncomprise administering irinotecan in a dose of about 0.1 mg/kgbodyweight to about 10 mg/kg bodyweight. In other embodiments,irinotecan may be administered in a dose of about 0.1, 0.2, 0.5, 1, 1.5,2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10mg/kg bodyweight, or more. In certain embodiments, an effective dose ofirinotecan is between about 0.5 mg/kg to about 9 mg/kg, between about 1mg/kg to about 8 mg/kg, between about 2 mg/kg to about 7 mg/kg, betweenabout 3 mg/kg to about 5 mg/kg.

When the composition of the present invention is formulated in unitdosage form, irinotecan will preferably be present in an amount ofbetween 10 mg and 1000 mg per unit dose. Preferably, the amount ofirinotecan per unit dose will be about 10, 15, 20, 25, 30, 35, 40, 45,50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 115, 120, 125, 130, 135,140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 210,220, 230, 240, 250, 260, 270, 280, 290, 300, 350, 400, 450, or 500 mg orany amount therein. In one embodiment, the amount of irinotecan per unitdose will be about 50 mg to about 350 mg, about 75 mg to about 250 mg,about 100 mg to about 200. In one embodiment, the unit dosage formcomprises 30, 45, 60, 75, 90, 120, 135, 150, or 180 mg of irinotecan.

In certain embodiments, the dosage of an N-oxide of vinca alkaloid oranalog thereof will be administered according to a metronomic dosing. Asused here, the term “metronomic dosing” refers to a dosing regimen,which uses a dose lower than the maximum tolerated doses (MTD) tominimize toxic side effects, administered at constant intervals withoutrest periods. For purposes of the present invention, the desiredpharmacological effect of metronomic dosing with an N-oxide of vincaalkaloid or analog thereof is inhibition of tumor growth. “Inhibition oftumor growth” means causing a suppression of tumor growth and/or causinga regression in tumor size. It is believed that metronomic dosingelicits repeated waves of apoptosis of tumor endothelial cells bytargeting cells of the vasculature which form the blood vessels of thetumor as opposed to the tumor cells themselves. Thus, metronomic dosingappears to abrogate tumor cells' apparent capability to repair andrecover during the usual rest periods between episodic application of acytotoxic drug at or near MTD, followed by periods of rest to allownormal tissues to recover.

The term “MTD” as used here for N-oxides of vinca alkaloids and analogsmay be identified as part of the clinical evaluation of the N-oxide. Forexample, phase I trials can include a determination of the maximumtolerated dose, dose-limiting toxicities (DLT) and pharmacokinetics ofan N-oxides of vinca alkaloids or analog. Thus, the MTD for any Food andDrug Administration (FDA) approved therapeutic compound can bedetermined by those of ordinary skill in the art. The MTD for anyparticular therapeutic compound may vary according to its formulation(e.g., injectable formulation, implantable bioerodible polymerformulation, oral formulation), route of delivery (e.g., intravenous,oral, intratumoral), manner of delivery (e.g., infusion, bolusinjection), dosing schedule (e.g., hourly, daily, weekly) and the like.MTD frequently is defined as the highest dose level at which 50 percentof subjects administered with the drug develop a dose-limiting toxicity.Other definitions which are clinically relevant and generally acceptedwill be known to one of ordinary skill in the art.

The present invention also provides metronomic therapy regimes. In someembodiments, there is provided a metronomic dosing of an N-oxide ofvinca alkaloid or analog, wherein the N-oxide is administered over aperiod of at least 1, 2, 3, 4, 5, 6, 7, or 8 weeks or 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 18, 24, 30, or 36 months wherein the interval betweeneach administration is no more than about 1, 2, 3, 4, 5, 6 or 7 days,and wherein the dose of the N-oxide of vinca alkaloid or analog at eachadministration is about 0.25% to about 80% of its MTD. In otherembodiments, the vinca alkaloid N-oxide is administered at no more than80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 23%, 20%,18%, 16%, 14%, 12%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of itsMTD, which is predicted to be 100 mg/m² to 700 mg/m². In someembodiments, there is provided a method of administering a compositioncomprising an N-oxide of vinca alkaloid or analog, wherein thecomposition is administered over a period of at least one week, whereinthe interval between each administration is no more than about a day,and wherein the dose of the N-oxide of vinca alkaloid or analog at eachadministration is about 0.25% to about 80% of its MTD or other dosesmentioned herein. In some embodiments, there is provided a method ofadministering a composition comprising an N-oxide of vinca alkaloid oranalog, wherein the composition is administered over a period of atleast one month, wherein the interval between each administration is nomore than about a week, and wherein the dose of the N-oxide at eachadministration is about 0.25% to about 80% of MTD or other dosesmentioned herein. In some embodiments, the composition is administeredat least about any of 1×, 2×, 3×, 4×, 5×, 6×, 7× (i.e., daily) a week.

In some embodiments, the compositions may be administered continuouslyover a pre-specified period, for example continuously transfusing thecompositions for about 0.25 to about 80% of the MTD or other dosesmentioned herein, wherein the pre-specified period is at least 1, 2, 3,4, 5, 6, 7, or 8 weeks. In some embodiments, the intervals betweensuccessive continuous administration (e.g., infusion) sessions are atleast about 1 day, 2 days, 3 days, 4 days, 5 days, six days, one week, 2weeks, 3 weeks, 4 weeks, or 5 weeks after which a new continuousadministration session is started.

In some embodiments, one or more patches applied at one or more parts ofthe body may be used to deliver a dose that does not exceed about 0.25to about 80% of the MTD of the N-oxide per application period, or otherdoses mentioned herein. In some embodiments, the intervals between eachadministration by patch are less than about any of 7 days, 6 days, 5days, 4 days, 3 days, 2 days, and 1 day. In some embodiments, thecomposition is administered by a patch over a period of at least aboutany of 1 day, 2 days, 3 days, 4 days, 5 days, six days, one week, 2weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, or 8 weeks. In someembodiments, the intervals between successive patch administrations areat least about 1 day, 2 days, 3 days, 4 days, 5 days, six days, oneweek, 2 weeks, 3 weeks, 4 weeks, or 5 weeks after which one or more newpatches are applied. Examples of transdermal patches that may besuitable to deliver metronomic doses of vinca alkoloid N-oxides andanalogs thereof may be found in U.S. Pat. Nos. 6,977,070, 7,022,340,6,004,581, 5,939,094, 5,624,677, 7,001,609, 6,632,522, 6,630,238,6,482,871, 6,086,911, 5,698,217, 5,639,469, 5,244,677, 4,451,260,6,173,851, 5,223,261, 5,192,548, 6,645,528, 5,700,480, 6,974,588,6,238,693, 6,000,548, 4,726,951, 4,721,619, 5,225,196, 4,983,392,6,103,257, 6,110,486, 5,955,098, 5,985,317, and 5,952,000.

In some embodiments, a controlled release formulation may be used tocontrol release rate of the N-oxide of vinca alkaloid or analog frompreparations so that a metronomic dosing regimen is maintained for apre-determined period of time. In some embodiments, the metronomicdosing of the N-oxide of vinca alkaloid or analog may be maintained byusing an erodable polymer matrix, reservoir device, microparticles,nanoparticles, osmotic pumps, or pH dependant coatings. In theseembodiments, the N-oxide of vinca alkaloid or analog may be delivered ata dose that does not exceed about 0.25 to about 80% of the MTD of theN-oxide per application period, or other doses mentioned herein.

In some embodiments, there is provided a metronomic dosing of an N-oxideof vinca alkaloid or analog, wherein the N-oxide is administered over aperiod of at least 1, 2, 3, 4, 5, 6, 7, or 8 weeks or 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 18, 24, 30, or 36 months, wherein the interval betweeneach administration is no more than about a week, and wherein the doseof the N-oxide at each administration is about 25 mg/m² to about 500mg/m². In some embodiments, there is provided a metronomic dosing of anN-oxide of vinca alkaloid or analog, wherein the N-oxide is administeredover a period of at least three months, wherein the interval betweeneach administration is no more than about a week, and wherein the doseof the N-oxide at each administration is about 25 mg/m² to about 500mg/m². In some embodiments, there is provided a metronomic dosing of anN-oxide of vinca alkaloid or analog, wherein the N-oxide is administeredover a period of at least one month, wherein the interval between eachadministration is no more than about one day, two days, three days, fourdays, five days, six days, or one week, and wherein the dose of theN-oxide at each administration is about 25 mg/m² to about 500 mg/m². Insome embodiments, there is provided a metronomic dosing of an N-oxide ofvinca alkaloid or analog, wherein the N-oxide is administered over aperiod of at least one week, wherein the interval between eachadministration is no more than about 12, 24, 36, 48, 60, 72, 84, 96,108, or 120 hours, and wherein the dose of the N-oxide at eachadministration is about 25 mg/m² to about 500 mg/m². In someembodiments, the dose of the N-oxide of vinca alkaloid or analog peradministration is less than about any of 25, 30, 35, 40, 45, 50, 55, 60,70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, and500 mg/m². In some embodiments, the composition is administered at leastabout any of 1×, 2×, 3×, 4×, 5×, 6×, and 7× (i.e., daily) a week. Insome embodiments, the intervals between each administration are lessthan about any of 7 days, 6 days, 5 days, 4 days, 3 days, 2 days, and 1day.

The relative proportion of ingredients in the compositions of theinvention will, of course, vary considerably depending on the particulartype of composition concerned. The relative proportions will also varydepending on the particular function of ingredients in the composition.The relative proportions will also vary depending on the particularingredients employed and the desired physical characteristics of theproduct composition, e.g., in the case of a composition for topical use,whether this is to be a free flowing liquid or a paste. Determination ofworkable proportions in any particular instance will generally be withinthe capability of a person of ordinary skill in the art. All indicatedproportions and relative weight ranges described below are accordinglyto be understood as being indicative individually inventive teachingsonly and not as limiting the invention in its broadest aspect.

The amount of compound having Formula I in compositions of the inventionwill of course vary, e.g., depending on the intended route ofadministration and to what extent other components are present. Ingeneral, however, the compound having Formula I will suitably be presentin an amount of from about 0.005% to 20% by weight based upon the totalweight of the composition. In certain embodiments, the compound havingFormula I is present in an amount of from about 0.01% to 15% by weightbased upon the total weight of the composition.

In addition to the foregoing, the present invention also provides aprocess for the production of a pharmaceutical composition ashereinbefore defined, which process comprises bringing the individualcomponents thereof into intimate admixture and, when required,compounding the obtained composition in unit dosage form, for examplefilling said composition into tablets, gelatin, e.g., soft or hardgelatin, capsules, or non-gelatin capsules.

The starting materials of the N-oxides of the present invention areknown and described, for example, in U.S. Pat. Nos. 6,555,547,6,365,735, RE37,449, 6,127,377, 5,369,111, 5,030,620, 5,024,835,4,831,038, 4,765,972, RE30,561 and 4,160,767.

In certain situations, more than one of the nitrogen atoms of the vincaalkaloid analog may be oxidized simultaneously. In certain cases, one ormore of the multiple N-oxide groups may be reduced selectively, leavingone or more of the other N-oxide groups in place. Thus, the presentinvention contemplates the preparation of N-oxide analogs in which oneor more of the nitrogen atoms that are suitable for N-oxide formationare present as the N-oxide without regard to the susceptibility of aparticular nitrogen atom to N-oxide formation or the susceptibility of aparticular N-oxide group to reduction. It is envisaged to employ acombination of suitable protecting groups (see: Greene, T. W.; Wuts, P.G. M. Protective Groups in Organic Synthesis, second edition, WileyInterscience, 1991) to protect those nitrogen atoms not undergoingoxidation.

By way of an example, primary and secondary amines that may be presentin a vinca alkaloid analog may be protected using, for example,tert-butyl sulfonyl (BUS) group. Jarowicki, K. and Kocienski, P., J.Chem. Soc., Perkin Trans 1, 4005-4037, 4029 (1998); Sun, P. and Weinreb,S. M. J. Org. Chem. 62:8604-08 (1997). The BUS protecting group isintroduced by reaction of the amine with tert-butylsulfinyl chloridefollowed by oxidation of the sulfinyl amide with, for example,dimethyldioxirane, m-chloroperbenzoic acid or RuCl₃ catalyzed NaIO₄. Theoxidation step in the preparation of the BUS-protected primary orsecondary amines may also oxide any tertiary amine and heteroaromaticnitrogen present in the compounds. Thus, this protecting group may beintroduced into primary and secondary amines while simultaneouslyoxidizing tertiary and heteroaromatic nitrogen atoms.

The BUS protecting group is stable towards strong reagents such asalkyllithium, Grignard reagents, 0.1M HCl in MeOH (20° C., 1 hr), 0.1MTFA in dichloromethane (20° C., 1 hr) and pyrolysis at 180° C. TheBUS-protected secondary amines can be cleaved with 0.1 M triflic acid indichloromethane containing anisole as a cation scavenger at 0° C. for15-30 minutes while primary amines are released more slowly at roomtemperature. If desired, both BUS-protected primary and secondary aminesmay be deprotected with 0.1 M triflic acid in dichloromethane containinganisole as a cation scavenger at 25° C. for 2.5 hours. Thus, the BUSprotecting group may allow protecting primary and secondary aminessimultaneously while also oxidizing tertiary amines and heteroaromaticnitrogen atoms to the N-oxides. Moreover, the BUS protecting group mayallow protecting primary and secondary amines simultaneously, oxidizingtertiary amines and heteroaromatic nitrogen atoms to N-oxides, deprotectthe secondary amine selectively, alkylate the secondary amine to atertiary amine, oxidize the resulting tertiary amine and deprotect theprimary amine. Alternatively, a primary and a secondary amine may beprotected with BUS protecting group, the secondary amine may bedeprotected selectively, the secondary amine may be protected with, forexample, Boc protecting group, and then the primary amine may bedeprotected selectively followed by alkylation and oxidation. Thus, whena primary amine and a secondary amine are present in a vinca alkaloidanalog, a BUS protecting group may be used to transform one of amines toan N-oxide without affecting the other.

Recent development in the use of Boc group to protect amines allowsintroduction and removal of the group under mild conditions. Forexample, a vinca alkaloid analog amine group may be protected with Bocgroup by simply mixing the analog and Boc-ON(2-(Boc-oxyimino)-2-phenylacetonitrile, available from Aldrich Co.) inbenzene at 25° C. for 20 minutes (or 6 hours if the amine is an electrondeficient aniline) in the presence of powdered zinc. See Spivey, A. C.and Maddaford A. Annu. Rep. Prog. Chem., Sect. B, 95:83-95 (1999). Alkylesters are tolerated.

Boc-protected amines are generally deprotected using triflic acidalthough recent developments generally use mildly acidic conditions thatleave acid-labile groups unaffected. For example, heating Boc protectedp-anisidines at 180° C. in the presence of 4-chlorophenol deprotects theamine group without affecting acid sensitive methoxy enols(—CH═C(OCH₃)—). Jarowicki, K. and Kocienski, P., J. Chem. Soc., PerkinTrans 1, 4005-4037, 4025 (1998). Thus, primary and secondary amines invinca alkaloid may be protected with Boc group followed by oxidation ofthe tertiary amines and deprotection of the primary and secondaryamines.

It has also recently been reported a new base-sensitive amino protectinggroup 1,1-dioxobenzo[b]thiophene-methoxycarbonyl (Bsmoc). Bsmoc isintroduced via its chloroformate or N-hydroxy-succinimide derivative.The Bsmoc group is stable towards tertiary amines for 24 hours but isremoved within 3-5 minutes using piperidine. Jarowicki, K. andKocienski, P., J. Chem. Soc., Perkin Trans 1, 4005-4037, 4027 (1998).Thus, primary and secondary amines present in vinca alkaloid analogs mayconveniently be protected with Bsmoc protecting group followed byoxidation of the tertiary amines and removal of the protecting groupunder mild conditions.

It has also been reported that certain heteroaromatic nitrogen atoms canbe oxidized selectively in the presence of certain aromatic primaryamines and certain secondary amines adjacent to a double bond. Delia, T.J. et al. J. Org. Chem. 30:2766-68 (1965). For example, oxidation ofcytosine with m-chloroperbenzoic acid results in cytosine 3-N-oxidedespite the presence of aromatic primary amine and a secondary amine.Thus, heteroaromatic nitrogen atoms and tertiary amines may be oxidizedin the presence of certain aromatic primary amines and secondary amines.

The following examples are illustrative, but not limiting, of the methodand compositions of the present invention. Other suitable modificationsand adaptations of the variety of conditions and parameters normallyencountered in clinical therapy and which are obvious to those skilledin the art are within the spirit and scope of the invention.

EXAMPLES

¹H NMR spectra were obtained from INOVA-300 MHz or INOVA-500 MHzspectrometers using D₂O or CDCl₃ as a solvent (s, d, t, dd and mindicate singlet, doublet, triplet, doublet of doublet, and multiplet,respectively). Analytical thin-layer chromatography was performed onpolyester-backed plates from EMD Chemicals, Inc., precoated with silicagel 60 F₂₅₄. Radial thin-layer chromatography was performed on aHarrison Research Chromatron (7924T) using 2 mm thick silica platescoated in our laboratory with silica gel 60 PF₂₅₄ containing gypsum (EMDChemicals, Inc.). Analytical scale high-performance liquidchromatography (HPLC) was performed on a 4.6 mm×250 mm MICROSORB C₁₈column using a pressure of 1800-2100 psi. The procedure for thesynthesis for the N-oxides of vinblastine, vincristine, and vinorelbinewas adapted from P. Mangeny et al. for the procedure for oxidation ofvinorelbine. The formation of vinblastine N-oxide leads to the formationof a new stereocenter. We have determined the configuration of the newlyintroduced oxygen atom in vinblastine N′_(b)-oxide synthesized below tobe cis to the nearby ethyl group by single crystal x-ray analysis of themaleate salt (FIG. 1). Both epimers of the N-oxide on either the R₈ orR₁₈ nitrogen atom in Formula I are contemplated in this invention.Vinblastine N-oxide is an intermediate for the synthesis of vinorelbine.See Mangeny, P. et al. Tetrahedron. 35:2175-2179 (1979).

Example 1 Synthesis of Vinblastine N′_(b)-Oxide Free Base and Salts:Sub-Gram Scale

Vinblastine free base. Vinblastine sulfate was purchased from AsianTalent. A 610-mg sample of vinblastine sulfate was dissolved in 20 mL ofdichloromethane and then it was shaken with 20 mL of 5% aqueous sodiumcarbonate. The organic layer was separated and aqueous solution wasextracted with dichloromethane (2×20 mL) and then with chloroform (20mL). The combined organic layer and extracts were dried over sodiumsulfate and evaporated to dryness, affording 526 mg (97%) of vinblastinefree base as a sticky white solid. The purity of vinblastine free basewas checked by ¹H 300 MHz NMR and HPLC (99.1% AUC) using a VarianCHROMSEP HPLC column SS 250×4.6 mm including holder with guard column,MICROSORB 100-5 C18; gradient: 30/70 (0.1% trifluoroacetic acid inacetonitrile)/(0.1% trifluoroacetic acid in water) to 100/0 (0.1%trifluoroacetic acid in acetonitrile)/(0.1% trifluoroacetic acid inwater); detection at 254 nm and 270 nm.

Vinblastine N-oxide free base. To a stirred solution of vinblastine freebase (526 mg, 0.65 mmol) in CH₂Cl₂ (5 mL) at 0° C. was added dropwise achilled solution of m-chloroperoxybenzoic acid (168 mg, 0.97 mmol) in 6mL of CH₂Cl₂. The progress of the reaction was followed by silica gelTLC (eluent: 5:0.7 CHCl₃-MeOH; vinblastine R_(f)=0.65; vinblastineN-oxide R_(f)=0.25). The reaction was complete after ˜2 min. Thesolution was washed with aqueous sodium carbonate (10 mL, 5 g/100 mL) toremove m-chlorobenzoic acid and any remaining m-chloroperoxybenzoicacid. The organic layer was dried over sodium sulfate and the solventwas evaporated under reduced pressure, yielding crude vinblastineN-oxide (600 mg). Chromatography over a short column of silica gel(eluent: 5:0.5 CHCl₃-MeOH) followed by radial chromatography (eluent:5:0.5 CHCl₃-MeOH) gave vinblastine N-oxide (340 mg, 64%) as a beigepowder. For larger scale preparations, the N-oxide may be obtained incomparable purity and yield by column chromatography over silica gel(eluent: 5:0.3 CHCl₃-MeOH to 5:0.7 CHCl₃-MeOH). The silica gel should bedeactivated with methanol prior to the chromatography. The 500 MHz ¹HNMR spectrum of vinblastine N′_(b)-oxide in CDCl₃ is consistent with theassigned structure. HPLC, >98% AUC.

Hydrogen Chloride Salt of Vinblastine N-oxide. A 55-mg (0.066 mmol)sample of vinblastine N-oxide was dissolved in 4 mL of methanol. Thesolution was cooled to −30° C. and then 33 μL (0.066 mmol) of 2N HCl inwater was added. The resulting solution was stirred for 5 min. Thensolvent was evaporated and the residue was dissolved in deionized water(4 mL), filtered and lyophilized to give the HCl salt of vinblastineN-oxide as a white somewhat hygroscopic powder. The 300 MHz ¹H NMRspectrum in D₂O is consistent with the assigned structure.

Maleic Acid salt of Vinblastine N-oxide. A 15-mg (0.018 mmol) sample ofvinblastine N-oxide was dissolved in 2 mL of methanol. To this solutionwas added 2 mg (0.018 mmol) of maleic acid dissolved in 0.170 mL ofmethanol at room temperature. The solution was stirred for 5 min andthen evaporated. Dichloromethane (DCM, 5 mL) was added to the residueand then the solution was concentrated to dryness to give 17 mg of themaleic acid salt of vinblastine N-oxide. The sample was dissolved in D₂O(0.7 mL) for an NMR spectrum, which was consistent with the assignedstructure. The D₂O solution was allowed to stand at room temperatureovernight. White plate-like crystals separated. Single crystal x-rayanalysis (FIG. 1) confirmed that the newly introduced oxygen atom isattached to the N′_(b) nitrogen atom and that this oxygen atom is cis tothe ethyl group at the 20′ position as seen in FIG. 1. The configurationof the N-oxide oxygen atom in vinblastine N′_(b)-oxide is the same asthat of the methiodide methyl group in leurocristine methiodidedehydrate as described in the x-ray crystal structure reported byMoncrief and Lipscomb (Moncrief, J. W.; Lipscomb, W. N. Acta Cryst.1966, 21, 322).

Example 2 Synthesis of Vinblastine N′_(b)-Oxide Free Base and Salts:Multi-Gram Scale

Vinblastine Free Base. A 3-L, three-neck, round-bottom flask equippedwith a mechanical stirrer, a nitrogen inlet, an addition funnel, and atemperature probe was purged with nitrogen. The flask was charged with asolution of vinblastine sulfate (40.0 g, 44 mmol, Guangzhou HuanyePharmaceutical Co.) in DCM (600 mL), followed by the addition of a 10%solution of Na₂CO₃ (140 mL, 132 mmol) at 0-5° C. over 10 min. Themixture was allowed to agitate at 0-5° C. for 20 min and then the layerswere separated after 5 min. The aqueous layer was extracted with DCM(2×200 mL) by agitating the mixture for 5 min and allowing phases toseparate for 5 min. Analysis of the aqueous layer after the extractionsdid not show any product. The combined organic extracts were washed withwater (200 mL) and dried over sodium sulfate. HPLC analysis of theorganic solution indicated 98.5% purity for the product. This solution(1.2 L) was diluted with DCM (0.6 L) and progressed to the oxidationstep without concentration.

Vinblastine N-Oxide free base. A 3-L, three-neck, round-bottom flaskequipped with a mechanical stirrer, a nitrogen inlet, an additionfunnel, and a temperature probe was purged with nitrogen. The flask wascharged with the solution of vinblastine free base in DCM (1.8 L),followed by the addition of a 70-75% solution of m-CPBA (10.6 g, 44mmol) in DCM (0.5 L) at −75 to −70° C. over 45 minutes. An in-processanalysis by TLC after 5 minutes indicated the full consumption ofvinblastine and formation of the N-oxide. The mixture was warmed to −10°C. and quenched with a 10% solution of Na₂CO₃ (200 mL) by agitating themixture at −10 to 0° C. for 5 minutes. The organic solution wasseparated after 5 minutes and washed with 10% Na₂CO₃ (200 mL), followedby water (200 mL). The solution was dried over sodium sulfate andconcentrated under vacuum. This afforded 40.2 grams of the crude productas a wet solid in 96.5% purity.

The crude N-oxide was dissolved in a 1:1 mixture of DCM/DMF (60 mL) anddiluted with MTBE (600 mL). The resultant slurry was agitated at ambienttemperature for 15 minutes. The solids were filtered and washed withMTBE (80 mL). The product was dried under vacuum at ambient temperaturefor 18 hours. This process yielded 37.3 g (102%) of the product in 97.8%purity as a white solid. Proton NMR spectrum of the solid was consistentwith the structure of the compound.

Vinblastine N-Oxide Dihydrochloride. A 1-L, three-neck, round-bottomflask equipped with a mechanical stirrer, a nitrogen inlet, an additionfunnel, and a temperature probe was purged with nitrogen. The flask wascharged with a solution of vinblastine N-oxide (37.3 g) in 2:1 isopropylalcohol (IPA)/MeOH (120 mL) and diluted with isopropyl acetate (IPAc,200 mL). The resultant solution was cooled to −35 to −30° C. A 4 Nsolution of HCl in IPA (21.1 mL, 84.4 mmol) was added over 10 min andthe mixture was allowed to stir at this temperature for 5 minutes. Themixture was then diluted with IPAc (1.4 L, 35 vol) and allowed to stirat 0-10° C. for 30 minutes. The solids were filtered and washed withIPAc (200 mL). HPLC analysis of the wet cake indicated 98.0% purity forthe product. The product was dried under vacuum at ambient temperatureand 29-30 in. Hg for 58 hours. This provided 35.4 g of the product as awhite solid. HPLC analysis indicated 97.7% purity for the solid. ¹H NMRspectrum was consistent with the structure of the compound but indicated7 wt % of residual IPAc, 1 wt % IPA, and 1 wt % DMF.

Diethyl Ether Slurry of Vinblastine N-Oxide Dihydrochloride Salt. Thebatch (33.3 g) was suspended in diethyl ether (500 mL, 15 vol) at 0-5°C. and agitated for 18 hours. The batch was filtered and the solids weredried at ambient temperature and 29-30 in. Hg for 24 hours. Thisprovided 29.9 g of the salt as a white solid. HPLC analysis indicated96.6% purity for the solid. ¹H NMR and mass spectra were consistent withthe structure of the compound but indicated ≈0.3 wt % diethyl ether.

Example 3 Synthesis of Vinorelbine N′_(b)-Oxide

Vinorelbine free base was purchased from Asia Talent. Purification, whennecessary, was accomplished by radial chromatography on silica gel using9:1 CHCl₃-MeOH as eluent.

Vinorelbine N′_(b)-oxide was synthesized using essentially the sameprocedure used to prepare vinblastine N′_(b)-oxide.m-Chloroperoxybenzoic acid (45 mg, 0.26 mmol) in CHCl₃ (2.5 mL) wasadded at 0° to a stirred solution of purified vinorelbine (150 mg, 0.20mmol) in CHCl₃ (4.5 mL) under nitrogen. After 15 min, the mixture waspoured into an aqueous sodium carbonate (27 mL, 40 g/L) and wasextracted by CHCl₃ (20 mL). After drying over sodium sulfate andfiltration, the CHCl₃ extract was evaporated under reduced pressureaffording crude vinorelbine N-oxide (125 mg, 83%). Thin-layerchromatography (silica; 9:1 CHCl₃/MeOH) indicated the presence of asmall amount of starting vinorelbine (vinorelbine R_(f)=0.37,vinorelbine N′_(b)-oxide R_(f)=0.18). Radial chromatography on silicagel (eluent, 9:1 CHCl₃/MeOH) gave vinorelbine N′_(b)-oxide as anoff-white solid (100 mg; 75%). The sample was dissolved in ethylacetate/hexanes and then the open flask was placed in a sealed containercontaining an open flask of hexanes. As the hexanes diffused slowly intothe vinorelbine N′_(b)-oxide solution, vinorelbine N′_(b)-oxideprecipitated out as an off-white powder (80 mg; 53%). The 300 MHz ¹HNMRspectrum was consistent with the assigned structure. The configurationof the N′_(b)-oxide oxygen atom is assigned the same stereochemistry asthat found in vinblastine N-oxide by x-ray crystallography.

Example 4a Synthesis of Vincristine N′_(b)-Oxide

Vincristine Free Base. Vincristine sulfate was purchased from AsianTalent. A 480-mg sample of vincristine sulfate was dissolved in 10 mL of7:3 chloroform/methanol and then it was shaken with a 15 mL of 5%aqueous sodium carbonate. The organic layer was separated and aqueoussolution was then extracted with dichloromethane (2×20 mL) followed bychloroform (2×20 mL). The combined organic layer and extracts were driedover sodium sulfate and concentrated to dryness, affording 420 mg (98%)of vincristine free base as a sticky yellowish-white solid. The purityof vincristine free base was checked by 1H 300 MHz NMR and HPLC (99.0%AUC).

Vincristine N′_(b)-Oxide. m-Chloroperoxybenzoic acid (132 mg, 0.76 mmol)in 2 mL of dichloromethane was added dropwise at 0° C. to a stirredsolution of vincristine free base (420 mg, 0.51 mmol) in dichloromethane(4 mL). The progress of the reaction was followed by silica gel TLC(eluent: 5:0.5 CHCl₃-MeOH; vincristine R_(f)=0.65; vincristine N-oxideR_(f)=0.30) and was complete after ˜2 min. The solution was washed withaqueous sodium carbonate (8 mL, 5 g/100 mL) to remove m-chlorobenzoicacid and any remaining m-chloroperoxybenzoic acid. The organic layer wasdried over sodium sulfate and the solvent was evaporated under reducedpressure, yielding crude vincristine N-oxide (450 mg). Chromatographyover a short column of silica gel (eluent: 5:0.5 CHCl₃-MeOH) followed byradial chromatography over silica gel (eluent: 5:0.5 CHCl₃-MeOH) gavevincristine N-oxide (288 mg, 67%) as an off-white powder. The 300 MHz ¹HNMR spectrum in CDCl₃ was consistent with the assigned structure.Reverse phase HPLC showed a purity of >98% (AUC). The configuration ofthe N-oxide oxygen atom is assigned the same stereochemistry as thatfound in vinblastine N-oxide by x-ray crystallography.

Example 4b Synthesis of Vincristine N′_(b)-Oxide

Vincristine Free Base. Vincristine sulfate was purchased from AsianTalent. A 970-mg sample of vincristine sulfate was dissolved in 20 mL of19:1 methylene chloride/methanol and then it was shaken with a 20 mL of5% aqueous sodium carbonate. The organic layer was separated and aqueoussolution was then extracted with dichloromethane (2×30 mL) followed bychloroform (1×30 mL). The combined organic layer was washed with waterand extracts were dried over sodium sulfate and concentrated to dryness,affording 870 mg (100%) of vincristine free base as a stickyyellowish-white solid. The purity of vincristine free base was checkedby ¹H 300 MHz NMR and HPLC (99.0% AUC).

Vincristine N′_(b)-Oxide. To a stirred solution of Vincristine free base(870 mg, 1.00 mmol) in CH₂Cl₂ (130 mL) at −78° C. a chilled (by standingon dry ice) solution of m chloroperbenzoic acid (66% m-CPBA, Aldrich,273 mg, 1.60 mmol) in 40 mL of CH₂Cl₂ was added dropwise by way of anunchilled separatory funnel during 30 minutes. The progress of thereaction was followed by TLC (eluent: CH₂Cl₂-MeOH, 5:0.8, R_(f)Vincristine 0.80, R_(f) Vincristine N-oxide 0.30). The reaction wascomplete after 10 minutes. The resulting solution was warmed to −10° C.and washed with aqueous Na₂CO₃ (3×10 mL, 5 g/100 mL) to remove excess ofm-CPBA and the resulting m-chlorobenzoic acid. The organic layer waswashed with water, dried over sodium sulfate and the solvent wasevaporated under reduced pressure in a 35° C. bath, yielding crudeVincristine N-oxide (about 900 mg). Column chromatography over silicagel (60-200 mesh, VWR International, Inc., in 100% CH₂Cl₂, eluent,CH₂Cl₂-MeOH 5:0.5 to 5:1) gave Vincristine N-oxide as an off white solid(670 mg, 74%). The silica gel (50 g of silica gel per 1 g of N-oxide)should be deactivated by washing with methylene chloride/methanol, 5:0.5mixture before using for the chromatography. The 300 MHz ¹H NMR spectrumin CDCl₃ was consistent with the assigned structure. Reverse phase HPLCshowed a purity of >99% (AUC).

Example 5 Cytotoxicity of Vinca-Alkaloids and Analog N-Oxides Thereof inLymphoma, Leukemia, Multiple Myeloma and Cells Derived from HemtalogicalMalignancies

The cytotoxicity of vinca alkaloids and analog N-oxides thereof ondifferent lymphoma, leukemia, and multiple myeloma cell lines will betested in vitro under normoxic as well as 0.2% O₂ hypoxic conditions.Standard viability assays using MTT or Alamar Blue dye will be conductedto determine the 50% Inhibitory Concentration (IC₅₀) for each compound.Cultured tumor cells will be treated with the compounds for 8 hr orgreater under normoxic or hypoxic conditions, and viability will bemeasured 24-48 hr later. In certain cases, enzyme inhibitors tobioreductive enzymes will be co-cultured with the cells to verifymechanism of action. Positive controls will use chemotherapeutic agentsat doses shown in the art to be effective. The results should indicatethat vinca alkaloids and its related N-oxide analogs are cytotoxic tomany of the cell lines derived from hematological malignancies, withIC₅₀ values in the nanomolar to sub-nanomolar range. Vinca alkaloidanalog N-oxides are expected to be increased or differentialcytotoxicity profiles than the parent compounds under normoxic or euoxicconditions.

Example 6 Anti-Tumor Activity of Vinca-Alkaloids and Analog N-OxidesThereof in Lymphoma, Leukemia, Multiple Myeloma and Cells Derived fromHemtalogical Malignancies

The in vivo antitumor efficacy of vinca alkaloids and analog N-oxideswill be evaluated using syngeneic and xenotransplant experimental murinemodels of hematological malignancies. For example, DBA/2 mice(n=12/group) will be inoculated intraperitoneally (ip) with 1×10⁵ murineL1210 or 1×10⁶ P388 leukemic tumor cells. Following tumor implantation,mice will be treated with 10-1000 mg/kg of vinca alkaloids and analogN-oxides by intravenous administration. Agents may be administered on asingle or multiple treatment regimen (ie. once weekly, q3d) to yieldoptimal dose schedule benefit. The effects of treatment with vincaalkaloids and N-oxide analogs compared to vehicle control treated miceon prolonging the survival of tumor bearing mice will be compared usinga pre-determined survival endpoint. Animals will also be monitored forsymptoms of acute toxicity such as body weight loss, neurotoxicities,lymphopenia and neutropenia. As a positive control, treatment will becompared with a standard agent.

In a related example, the in vivo antitumor efficacy of vinca alkaloidsand analog N-oxides will be evaluated using a Namalwa human lymphomaxenograft model in nude mice. Female nu/nu mice (n=10/group) will beimplanted with human Namalwa lymphoma cells by subcutaneous injection.Pair-matched mice will be randomized to different treatment groups whentheir tumors are approximately 50-100 mm³ in size as determined bycaliper measurements. Mice will be treated with 10-1000 mg/kg of vincaalkaloids and analog N-oxides by intravenous administration on a weeklyor once every 3 day schedule. The anti-tumor effects of the compoundswill be assessed as tumor growth inhibition (TGI) and tumor growth delay(TGD) by established criteria and practices known in the art. Treatmenteffects will also be compared to a standard agent known in the art suchas mitoxantrone or doxorubicin.

In a related example, the in vivo antitumor efficacy of vinca alkaloidsand analog N-oxides will be demonstrated in a human xenotransplantionmodel of acute lymphocytic leukemia (ALL) in immunodeficient mice.NOD/SCID mice will be implanted with primary human ALL tumor cells.Tumor grafting and burden will be monitored by flow cytometry usingstandard leukemia identification markers and pair-matched animals willbe randomized into control or treatment groups. Seven to 10 daysfollowing transplant, mice will be injected intravenously (iv) with10-1000 mg/kg of vinca alkaloids and analog N-oxides by intravenousadministration on a weekly or once every 3 day schedule. Mice will bebled routinely and assessed for tumor burden by FACS analysis. Thenumber of leukemic tumor cells per ml of blood prior and post-treatmentwill be compared to evaluate compound efficacy. Animals will also bemonitored for symptoms of acute toxicity such as body weight loss,neurotoxicities, lymphopenia and neutropenia. As a positive control,treatment will be compared with a standard agent.

Further, the in vivo antitumor activity of vinca alkaloids and analogN-oxide in combination with chemotherapeutic agents and/or radiotherapywill be evaluated using a xenograft model in nude mice.

Further, the in vivo antitumor activity of vinca alkaloids and analogN-oxide in combination with therapeutic monoclonal antibodies such asanti-CD52 (Campath), anti-CD20 (Rituxan, Zevalin, Bexxar), anti-CD22(LymphoCide) anti-CD33 (MyloTarg), or HLA-DR (Lym-1, Oncolym) will beevaluated using a xenograft model in nude mice.

Further, the in vivo antitumor activity of vinca alkaloids and analogN-oxide in combination with small molecular weight inhibitors of kinasessuch as imatanib (Gleevec) will be evaluated using a xenograft model innude mice.

Example 7 Cytotoxicity of Vinca-Alkaloid Analogs and N-Oxides Thereof inSolid Tumor Lines

The differential cytotoxicity of vinca-alkaloids and analog N-oxidesthereof on different solid tumor cell lines are demonstrated in vitrounder normoxic conditions and 0.2% O₂ hypoxic conditions. Shown in FIGS.2A-2D and 3A-3D and Tables 2 and 3 are results obtained from treatmenteffects of vinblastine N-oxide and vincristine N-oxide, against humanH460 lung adenocarcinoma, HT29 colorectal adenocarcinoma, A549 non-smallcell lung carcinoma, FaDU head and neck tumor cell lines in vitro. Tumorcell line were cultured in DMEM-10% FBS under standard conditions.One-hundred mm plastic culture dishes were seeded with 1×10⁶ tumor cellsand treated with vinca alkaloids or N-oxide analogs at a dose range of0.01-100 nM. Cells were exposed with gentle rocking to a constant levelof low oxygen (0.2% O₂-5% CO₂-balance N₂) in a hypoxia apparatus(INVIVO2400 Hypoxia Workstation, Ruskin Technology) for 14 hr at 37° C.Identically treated cells were incubated under normoxia (air-5% CO₂) at37° C. Cells were harvested and replated in fresh medium at a density of1×10³ cells/well in 48-well plastic culture plates to assess viability24-72 hr later. Briefly, Alamar Blue was added to replicate wells on Day0 and Day 3, and cells were incubated for a further 3-6 hr at 37° C.before fluorescence readings using a plate reader (530-560 nmexcitation, 590 emission). The percent inhibition of proliferation wascalculated and plotted against control-treated cells at each drugconcentration. The 50% growth inhibitory concentration (IC₅₀) valueswere calculated for the paired normoxic and hypoxic treatments. TheHypoxia Cytotoxicity Ratio (HCR) was determined as the ratio of the IC₅₀of the compound under normoxic conditions vs. hypoxic conditions.

As shown in FIGS. 2A-2D and Table 2, it was demonstrated thatvinblastine N-oxide has decreased cytotoxic activity under normoxicconditions compared to the parent vinblastine compound against multiplehuman solid tumor cell lines including H460 lung, HT29 colorectal andA549 lung adenocarcinoma cells. Upon hypoxia exposure, vinblastineN-oxide treatment was demonstrated to have potent cytotoxic activitywith comparable activity to vinblastine as shown in FIG. 2D and Table 2.The HCR values of 3-21 for vinblastine N-oxide as shown in Table 2demonstrates hypoxia-induced cytotoxicity of this compound againstmultiple human solid tumor cell line in vitro.

TABLE 2 Cytotoxicity and 50% inhibitory concentrations (IC50) ofvinblastine N- oxide or vinblastine on human tumor cell lines exposed tonormoxic vs. hypoxic conditions. Hypoxia Cell IC50 Air IC50 HypoxiaCytotoxicity Ratio Compound Line (mM) (mM) (HCR)* Vinblastine H460 0.150.07 21.4 N-oxide H460 0.2 0.02 10 H460 0.2 0.03 6.7 Vinblastine H4600.02 0.017 1 Vinblastine A549 0.15 0.035 4.3 N-oxide A549 0.45 0.10 4.5Vinblastine HT29 0.15 0.05 3 N-oxide HT29 0.2 0.07 2.9 Vinblastine SiHa4 0.7 4.3 N-oxide Vinblastine FaDu 0.15 0.02 7.5 N-oxide *HCR is definedas (IC50 air)/(IC50 hypoxia)

As shown in FIGS. 3A-3D and Table 3, it was demonstrated thatvincristine N-oxide has decreased cytotoxic activity under normoxicconditions compared to the parent vincristine compound against multiplehuman solid tumor cell lines. Upon hypoxia exposure, vincristine N-oxidetreatment was demonstrated to have potent cytotoxic activity withcomparable activity to vincristine (FIG. 3D and Table 3). The HCR valuesof 5-25 for vincristine N-oxide as shown in Table 3 demonstrateshypoxia-induced cytotoxicity of this compound against multiple humansolid tumor cell line in vitro.

TABLE 3 Cytotoxicity and 50% inhibitory concentrations (IC50) ofvincristine N- oxide or vincristine on human tumor cell lines exposed tonormoxic vs. hypoxic conditions. Hypoxia Cell IC50 Air IC50 HypoxiaCytotoxicity Ratio Compound Line (mM) (mM) (HCR)* Vincristine N- H4601.5 0.06 25 oxide H460 0.9 0.065 14 H460 1 0.2 5 Vincristine H460 0.0170.015 1.1 Vincristine N- A549 0.9 0.08 11.2 oxide Vincristine N- FaDu0.09 0.015 6 oxide *HCR is defined as (IC50 air)/(IC50 hypoxia)

Example 8 Cytotoxicity of Vinca Alkaloid Analogs and N-Oxides Thereof inSolid Tumor Lines Using Clonogenic Assays

The differential cytotoxicity of vinca alkaloids and analog N-oxidesthereof on different solid tumor cell lines are demonstrated in vitrounder normoxic conditions and 0.2% O₂ hypoxic conditions using colonyformation (clonogenic) assays. Shown are results in FIG. 4 obtained fromtreatment effects of vinblastine N-oxide on the growth of viablecolonies of H460 lung carcinoma tumor cells following exposure tonormoxic (20% O₂) vs. hypoxic (0.2% O₂) conditions.

Human H460 lung adenocarcinoma tumor cells were cultured in DMEM-10% FBSunder standard conditions and seeded in 100 mm plastic culture dishes ata density of 1×10⁶ cells/dish. Cells were then treated with vinblastineN-oxide at a 0.01-100 nM dose range. Cells were exposed with gentlerocking to a constant level of low oxygen (0.2% O₂-5% CO₂-balance N₂) ina hypoxia apparatus (INVIVO2400 Hypoxia Workstation, Ruskin Technology)for 14 hr at 37° C. Identically treated cells were incubated undernormoxia (air-5% CO₂) at 37° C. Cells were harvested and replated infresh medium at a density of 1×10⁵ cells/well in fresh plastic cultureplates to assess the growth of viable colonies 7 days later. Colonieswere fixed, stained with 0.4% crystal violet and enumerated 7 dayslater. The 50% Inhibitory concentration to block the number of viablecolonies compared to control-treated cells under normoxic and hypoxicconditions are calculated. As shown in FIG. 4, vinblastine N-oxide haddecreased cytotoxic activity against H460 lung tumor cells undernormoxic conditions compared to its potent cytotoxic activity underhypoxic conditions. Vinblastine N-oxide demonstrates a HypoxiaCytotoxicity Ratio of ˜20 against the growth of viable H460 tumorcolonies in vitro (FIG. 4). The results demonstrated that vinblastineN-oxide is a prodrug that is activated under hypoxia to a potentcytotoxin with low nanomolar IC₅₀ inhibitory activity against humansolid tumor cell lines in vitro.

Example 9 Activation of Cytotoxicity of Vinca Alkaloid N-Oxides AnalogsAgainst Human Solid Tumor Cell Lines is Oxygen Dependent

Our results demonstrate the vinca alkaloid N-oxide analogs havedecreased cytotoxic activity under normoxic conditions but are activatedto potent cytotoxic agents under conditions of hypoxia. Human H460 lungadenocarcinoma tumor cells were cultured in DMEM-10% FBS under standardconditions. One-hundred mm plastic culture dishes were seeded with 1×10⁶tumor cells/dish and treated with vinca alkaloids or N-oxide analogs ata 0.01-100 nM dose range. Cells were exposed with gentle rocking to aconstant level of low oxygen (0.2, 1% or 5% O₂-5% CO₂-balance N₂) in ahypoxia apparatus (INVIVO2400 Hypoxia Workstation, Ruskin Technology)for 14 hr at 37° C. Identically treated cells were incubated undernormoxia (air-5% CO₂) at 37° C. Cells were harvested and replated infresh medium at a density of 1×10³ cells/well in 48-well plastic cultureplates to assess viability 24-72 hr later. Briefly, Alamar Blue wasadded to replicate wells on Day 0 and Day 3, and cells were incubatedfor a further 3-6 hr at 37° C. before fluorescence readings using aplate reader (530-560 nm excitation, 590 emission). The percentinhibition of proliferation was calculated and plotted againstcontrol-treated cells at each drug concentration. The 50% growthinhibitory concentration (IC₅₀) values were calculated for the pairednormoxic and hypoxic treatments. The Hypoxia Cytotoxicity Ratio (HCR)was defined as ((IC₅₀ in Hypoxia)/(IC₅₀ in Normoxia)). In FIG. 5 wedemonstrate that the activation of cytotoxicity against human H460 lungadenocarcinoma cells is oxygen dependent. Increased cytotoxicity ofvinblastine N-oxide against cultured H460 lung tumor cells was observedas the oxygen concentrations were decreased. Shown in FIG. 5 are theHypoxia Cytotoxicity Ratio (HCR) of vinblastine N-oxide plotted againstthe partial O₂ concentrations of 0.2%, 1%, and 5% O₂ that were tested.

Example 10 Bioreduction of Vinca-Alkaloid N-Oxides to Their RespectiveParent Compounds in Hypoxic Cancer Cells as Measured by LC/MS-MSAnalysis

Here we demonstrate that vinblastine N-oxide and vicristine N-oxide arebioreduced under (0.2% O₂) hypoxic conditions to their respective parentcompounds as detected by chromatography and mass spectrometric analysis.Human H460 lung adenocarcinoma tumor cells were seeded in 100 mm dishesat a density of 1×10⁶ cells/dish and untreated or treated with vincaalkaloid N-oxide analogs at a dose range of 0.02-7 μM. Cells wereexposed with gentle rocking to a constant level of low oxygen (0.2%O₂-5% CO₂-balance N2) in a hypoxia apparatus (INVIVO2400 HypoxiaWorkstation, Ruskin Technology) for 12 hr at 37° C. Identically treatedcells were incubated under normoxia (air-5% CO₂) at 37° C. Cells wereharvested and lysed in DNA lysis buffer (20 mM Tris-HCL, pH 8.0, 1 mMEDTA, 0.1% Triton X-100) and stored at −80° C. for analysis of DNAcontent and LC/MS/MS analysis. Lysates were thawed and analysed for DNAusing Hoescht 33258dye and fluorescence (λ_(excitation) 350 nm,λ_(emmision) 455 nm) in order to normalize the measured vinca alkaloidor N-oxide analog concentrations to amounts of cells lysed in theexperiment. Lysates were mixed with 150 ml of methanol containing 0.1%acetic acid and vinca alkaloid internal standard, and vortexed for 10min, and centrifuged for 5 min at 18000 g. Clarified supernatants weretransferred to HPLC vials fitted with glass inserts and analyzed byLC-MS/MS. Chromatography was performed on an Acquity HPLC system fittedwith an Acquity BEH C18 column, and used 0.1% (v:v) formic acid in waterfor mobile phase A, and 0.1% (v:v) formic acid in acetonitrile formobile phase B. Mass spectral analysis was performed using a MicromassQuattro Micro triple quadupole mass spectrometer.

In FIGS. 6A-6D are shown the chromatograms of vinblastine andvinblastine N-oxide analog of the HPLC/MS-MS analysis of extracellularmedium from vinblastine N-oxide treated H460 tumor cells under normoxiaand hypoxia conditions. The vinblastine chromatogram distinctlyillustrates the increased liberation of vinblastine and a decreased peakof vinblastine N-oxide under hypoxia exposure. In FIGS. 7A and 7B areshown the quantitative amounts of vinblastine N-oxide and thevinblastine parent compound measured using mass spectral analysis fromlysates or the extracellular medium of 0.2 μM vinblastine N-oxidetreated cells under hypoxic or normoxic conditions. The amounts of eachcompound were calculated from standard curves. These results clearlydemonstrate that vinblastine N-oxide is bioreduced to vinblastine underhypoxia exposure and is detected in both the lysates and extracellularmedium of treated cells. The extracellular liberation of vinblastineupon hypoxia activation in treated cells indicates that vinblastineN-oxide analog may have potential bystander cytotoxic effects.

In FIGS. 8A and 8B are shown the quantitative amounts of vincristineN-oxide and the vincristine parent compound measured using mass spectralanalysis from lysates or the extracellular medium of 7 μM vincristineN-oxide treated H460 lung adenocarcinoma cells under hypoxic or normoxicconditions. The amounts for each compound were calculated from standardcurves. These results clearly demonstrate that vincristine N-oxide isbioreduced to vincristine under hypoxia exposure and is detected in boththe lysates and extracellular medium of treated cells. The extracellularliberation of vincristine upon hypoxia activation in treated cellsindicates that vincristine N-oxide analog may have potential bystandercytotoxic effects.

Example 11 Vinca-Alkaloid N-Oxide Analogs have Reduced Systemic andLethal Toxicity Compared to Their Parental Vinca-alkaloids In Vivo

We demonstrate here that vinblastine N-oxide and vincristine N-oxideanalogs have decreased systemic and lethal toxicity compared to theirrespective parent compounds, vinblastine and vincristine, in rodents invivo.

In FIG. 9 are shown the results of a 28-day acute toxicity study offemale athymic nu/nu mice that were injected intraperitoneally (ip) with6-60 mg/kg vinblastine N-oxide once every three days for a total of 5treatments (q3d×5). Body weights were monitored twice weekly and up to 2weeks following treatment. Mice were also injected ip with 4 or 6 mg/kgvinblastine using the same q3d×5 schedule as a positive control. Shownin FIG. 9 are the % Body Weight Loss of mice (n=5/group) treated withvinblastine N-oxide at a dose range of 6-60 mg/kg or vinblastine at 4-6mg/kg. Mice treated with vinblastine at a lethal dose of 4 or 6 mg/kgsuccumbed with a median day of death of 10.8 or 9.8 days, respectively.In contrast, mice treated with vinblastine N-oxide at doses of up to 15times the lethal dose of vinblastine showed no evidence of significantbody weight loss or gross symptoms of toxicity at 14 days following thelast treatment.

In FIG. 10 are shown the results of a 28-day acute toxicity study offemale athymic nu/nu tumor-bearing mice that were injected intravenously(iv) with 0.6-10 mg/kg vincristine N-oxide once every three days for atotal of 5 treatments (q3d×5). Body weights were monitored twice weeklyand up to 2 weeks following treatment. Mice were also injected iv with0.6 or 1 mg/kg vincristine using the same q3d×5 schedule as a positivecontrol. Shown in FIG. 10 are the % Body Weight Loss of mice (n=5/group)treated with vincristine N-oxide at a dose range of 0.6-10 mg/kg orvincristine at 0.6-1 mg/kg. Mice treated with vincristine showedsignificant body weight loss during the course of treatment. Incontrast, mice treated with vincristine N-oxide at doses of up to 10times the maximum tolerated dose of vincristine showed no evidence ofsignificant body weight loss or gross symptoms of toxicity at 14 daysfollowing the last treatment.

Example 12 Anti-Tumor Activity of Vinca-Alkaloids and Analog N-OxidesThereof in Solid Tumor Models in Mice

The in vivo antitumor efficacy of vinca-alkaloid and analog N-oxideswill be evaluated using xenograft murine models. For example, human H460lung adenocarcinoma, HT29 colorectal adenocarcinoma, or A549 lungcarcinoma tumor fragments (approximately 1 mm³) will be implantedsubcutaneously (sc) into female nude (nu/nu) mice. When the tumors reachapproximately 100 mm³ in size (25 days following implantation), theanimals will be pair-matched into 10 mice per group. Mice will beinjected iv with vinca-alkaloid N-oxide analog at a dose of 1-200 mg/kgon a q3d×5 schedule. Mice will also be treated with vehicle as anegative control and the bioreduced parent compound at the maximaltolerated dose as a positive control using the same schedule. Tumorswill be measured with calipers twice weekly and tumor volume calculatedusing the formula: Tumor Volume (mm³)=((width)²×length)/2. Animals willbe monitored for signs of toxicity and weighed daily for the first 5days of the study and then twice-weekly until the study end. Aspre-defined in the protocol, each animal will be actively euthanizedwhen its tumor reached the pre-determined endpoint size of 1200 mm³ orat the conclusion of the study (day 60) whichever comes first. The timeto endpoint (TTE) will be calculated for each mouse as TTE(days)=(log₁₀(endpoint volume mm³)−b)/m where b is the intercept and mis the slope of the line obtained by linear regression of alog-transformed tumor growth data set. Treatment outcome will bedetermined from tumor growth delay (TGD) which is defined as theincrease in median TTE in a treatment (T) group as compared to thecontrol (C) group (TGD=T−C) expressed in days or as a percentage of themedian TTE of the control group % TGD=((T−C)/C)×100. Kaplan-Meier plotswill be constructed to demonstrate the percentage animals remaining inthe study as a function of time following treatment. Statisticalsignificance between the treated vs. control groups will be evaluated bylogrank analysis.

In another example, female nude mice (nu/nu) will be implanted sc withfragments of human BxPC-3 pancreatic tumors. When the tumors reachapproximately 60-80 mm³ in size, the animals will be pair-matched intotreatment and control groups containing ten mice per group. Mice will beinjected iv with vinca-alkaloid N-oxide analog at a dose of 1-200 mg/kgon a q3d×5 schedule. Mice will also be treated with vehicle as anegative control and the bioreduced parent compound at the maximaltolerated dose as a positive control using the same schedule. Tumorswill be measured with calipers twice weekly and tumor volume calculatedusing the formula: Tumor Volume (mm³)=((width)²×length)/2. Animals willbe monitored for signs of toxicity and weighed daily for the first 5days of the study and then twice-weekly until the study end. Aspre-defined in the protocol, the experiment will be terminated when thevehicle control group tumor size reaches an average of ˜2000 mm³ (˜27days). Upon termination, the mice will be weighed, sacrificed, tumorsexcised, and the mean tumor volume per group calculated. Tumor growthinhibition (TGI), defined as the change in mean tumor volume of thetreated groups/the change in mean tumor volume of the control group×100(ΔT/ΔC) will be calculated for each group. Statistical comparisons willbe carried out using ANOVA followed by the Dunnett multiple comparisonstest.

The anti-tumor activity of vinca-alkaloid N-oxides will also be assessedin an orthotopic (ot) solid tumor xenograft model. In one embodiment,female athymic mice will injected with 2.5×10⁶ human BxPC-3 pancreaticcells directly into the pancreas parenchyma. Treatment with vehicle(negative control), 10-200 mg/kg vinca-alkaloid N-oxide analog (iv), or40 mg/kg gemcitabine (ip) will be initiated on day 14 post-tumorimplantation at a q3d×5 schedule. Treatment groups will consist of 12mice/group; an extra 5 mice be added to each group for histologicalanalysis. Mice will be monitored twice daily for symptoms of disease andactively euthanized based on the criteria outlined by the InstitutionalEthical Committee; the day of sacrifice will be considered the day ofcancer death. Prolongation of survival, a primary endpoint in the study,will be evaluated using Kaplan-Meier plots and statistical significanceof treatment responses compared to control groups will be analyzed bylogrank analysis. For histological analysis, mice will be sacrificed atmultiple days throughout treatment course and blinded tissue sectionsfrom resected tumors will be analysed by standard immunohistochemicaltechniques for detection of tumor proliferation (BrDUrd incorporation),hypoxia (pimonidazole staining), vasculature (CD31 staining), apoptosis(TUNEL staining) and necrosis. The effects of vinca-alkaloid N-oxideanalogs in comparison to vehicle control treated on the tumormicroenvironment will be qualitatively and quantitatively assessed. Inaddition, other target organs such as the lung and liver thatdemonstrate metastases formation will be resected and analysed fortreatment effects on metastatic spread. Five μm-thick formalin-fixedparaffin-embedded tissue sections will be stained with hematoxylin andeosin (H&E) gross histopathological analysis will be performed in ablinded fashion to compute the metastatic tumor burden. The totalpercentage of space occupied by invasive tumor cells was enumerated andwill be expressed as a percentage of total tissue evaluated. Statisticalanalysis will be computed using the non-parametric Mann-Whitney U-test.The effects of vinca-alkaloid N-oxides on preventing the growth ofmetastatic tumor cells and preventing the metastatic spread of hypoxictumor cells will be evaluated.

Further, the in vivo antitumor activity of vinca-alkaloid and analogN-oxide in combination with chemotherapeutic agents and/or radiotherapywill be evaluated using a xenograft model in nude mice.

Further, the in vivo antitumor activity of vinca-alkaloid and analogN-oxide in combination with biological agents, such as therapeuticmonoclonal antibodies (anti-VEGF, anti-EGF) will be evaluated using axenograft model in nude mice.

Further, the in vivo antitumor activity of vinca-alkaloid and analogN-oxide in combination with small molecules tyrosine kinase inhibitorswill be evaluated using a xenograft model in nude mice.

Further, the in vivo antitumor activity of vinca-alkaloid and analogN-oxide in combination with vascular disrupting, vascular damaging, oranti-angiogenic agents will be evaluated using a xenograft model in nudemice.

Further, the in vivo antitumor activity of vinca-alkaloid and analogN-oxide in combination with Hypoxia-Inducible Factor-1 or -2 (HIF-1 orHIF-2) antagonists will be evaluated using a xenograft model in nudemice.

Further, the in vivo antitumor activity of vinca-alkaloid and analogN-oxide in combination with other therapeutic agents that target euoxictumor cells will be evaluated using a xenograft model in nude mice.

Further, the in vivo antitumor activity of vinca-alkaloid and analogN-oxide in combination with other agents that decrease tumor oxygenationor increase tumor consumption will be evaluated using a xenograft modelin nude mice.

Example 13 Tumor Selectivity of Vinca-Alkaloids N-Oxide Analogs

The systemic and local tumor concentrations of vinca-alkaloid N-oxideanalogs and their bioreduced parent compound metabolites will bemonitored in tumor bearing mice using quantitative analytical proceduressuch as HPLC/MS-MS.

In one example, female athymic nude mice will be sc injected with 1×10⁷H460 lung adenocarcinoma tumor cells. Animals will be dosed when themean tumor volume reached 300 mm³. Mice will be randomized intotreatment groups of 5 mice/group. The mice will receive a single ivbolus injection of vinca alkaloid N-oxide at 10-200 mg/kg by tail-veininjections. Three mice per dose will be sacrificed at 15 min, 30 min, 1hr, 2 hr, 4 hr, 8 hr, 12 hr and 24 hr post-treatment. Plasma and driedtumor samples will be collected and stored at −80° C. for furtheranalysis using HPLC/MS/MS. Quantitation of vinca-alkaloid N-oxides andits bioreduced metabolites, from plasma and tumor samples will bedetermined following liquid-liquid extraction in methanol containing0.1% (v/v) acetic acid. Following liquid extraction, plasma samples willbe spiked with known concentrations of the internal standard, andsubjected to centrifugation at 15000×rpm for 10 min at 4° C. Theresulting supernatants will be analyzed by chromatography using anAcquity HPLC system fitted with an Acquity BEH C18 column, and used 0.1%(v:v) formic acid in water for mobile phase A, and 0.1% (v:v) formicacid in acetonitrile for mobile phase B. Mass spectral analysis will beperformed using a Micromass Quattro Micro triple quadupole massspectrometer. Resected tumor samples (˜300 mg) will be homogenized incold water, spiked with known internal standard and subjected toliquid-liquid extraction. Following the centrifugation of tissuehomogenates, the supernatants will be collected and concentrated to afinal volume of 0.4 ml using a vacuum concentrator prior to HPLC/MS/MSanalysis as described above. The amounts of vinca-alkaloid N-oxides andits bioreduced parent metabolite will be analysed from plasma and tumorsamples at various times post-treatment to demonstrate increased tumorselectivity and decreased systemic exposure following drugadministration.

Example 14 In Vivo Anti-Tumor Efficacy of Vinca-Alkaloids and AnalogN-Oxides Thereof in Murine Syngeneic Leukemias

The in vivo antitumor efficacy of vinca alkaloids and analog N-oxideswere evaluated using syngeneic experimental murine models ofhematological malignancies. For example, B6D2F1 mice (n=8/group) wereinoculated subcutaneously (sc) in the right flank with 1×10⁶ murineL1210 leukemic tumor cells. Growth of tumors was monitored as theaverage tumor size reached 80 to 120 mm³. Seven days later (day 1 of thestudy) mice were pair matched into the appropriate treatment groups andwere treated with vincristine, vinblastine and analog N-oxides byintravenous administration via the tail vein. Test agents wereadministered on multiple treatment regimen (q3d×5) to yield optimal doseschedule benefit. Vehicle control treated mice served as the negativecontrol group; mice treated with the bioreduced parent compounds,vincristine and vinblastine, dosed at their mean therapeutic doses(MTDs), served as the positive control groups. Tumors were measured withcalipers twice weekly and tumor volume calculated using the formula:Tumor Volume (mm³)=((width)²×length)/2. Animals were monitored for signsof toxicity and weighed daily for the first 5 days of the study and thentwice-weekly until the study end. As pre-defined in the protocol, eachanimal was actively euthanized when its tumor reached the pre-determinedendpoint size of 2000 mm³ or at the conclusion of the study (day 37)whichever came first. The time to endpoint (TTE) was calculated for eachmouse as TTE (days)=(log₁₀(endpoint volume mm³)−b)/m where b is theintercept and m is the slope of the line obtained by linear regressionof a log-transformed tumor growth data set. Treatment outcome wasdetermined from tumor growth delay (TGD) which is defined as theincrease in median TTE in a treatment (T) group as compared to thecontrol (C) group (TGD=T−C) expressed in days or as a percentage of themedian TTE of the control group % TGD=((T−C)/C)×100. Kaplan-Meier plotswere constructed to demonstrate the percentage animals remaining in thestudy as a function of time following treatment. Statisticalsignificance between the treated vs. control groups was evaluated bylogrank analysis.

As indicated in FIG. 11, VCR-NO resulted in a significant dose dependentL1210 tumor growth delay with superior activity as compared to thevincristine parent compound. Administered at a 75 mg/kg dose on a q3d×5schedule, VCR-NO resulted in 49% TGD (P<0.01) as compared to 8% forvincristine (NS). Similarly, using survival as an endpoint (FIG. 12),VCR-NO dosed at 75 mg/kg q3d×5 resulted in 28.2 days median survival ascompared with 19.6 days for vincristine given at its MTD of 1 mg/kgq3d×5 and 18.1 days for vehicle treated mice.

As shown in FIGS. 13 and 14 VBL-NO also demonstrated significantanti-tumor efficacy in the L1210 leukemia model. VBL-NO dosed at 30mg/kg on a q3d×5 schedule resulted in 41% TGD with a median survival of25.5 days as compared to 18.1 days for vehicle treated mice. The antitumor efficacy seen with VBL-NO dosed at 30 mg/kg q3d×5 was comparableto the parental compound vinblastine given at its MTD of 5 mg/kg q3d×5(34% TGD, 24.3 days survival).

In a related example, the in vivo antitumor efficacy of vinca alkaloidsand analog N-oxides was evaluated using another syngeneic experimentalmurine model of hematological malignancies, P388. This model wasdeveloped by subcutaneously inoculating B6D2F1 mice (n=8/group) with1×10⁶ murine P388 leukemic tumor cells using similar methodology to thatdescribed above for L1210. Seven days after tumor inoculation, when theaverage tumor size reached 80 to 120 mm³ (day 1 of the study), mice wereplaced into the appropriate treatment groups and were treated with theparental agents, vincristine and vinblastine, as well as the testagents, the analog N-oxides, by intravenous administration. Agents wereadministered on multiple treatment regimen (q3d×5) to yield optimal doseschedule benefit. Efficacy of treatment with the N-oxide analogs wasdetermined by Tumor Growth Delay (TGD) and Kaplan-Meier survivalanalysis as described for L1210, the exception being that the conclusionof the study was day 45 or the predefined endpoint size of 2000 mm³whichever came first. Vehicle control treated mice served as thenegative control group; mice treated with the standard agents,vincristine and vinblastine, dosed at their MTDs, served as the positivecontrol groups.

As indicated in FIG. 15, VCR-NO resulted in a highly significant P388tumor growth delay. Administered at a 60 mg/kg dose on a q3d×5 schedule,VCR-NO resulted in 76% TGD (P<0.001) as compared to 54% for vincristine.Similarly, using survival as an endpoint (FIG. 16) VCR-NO dosed at 60mg/kg q3d×5 resulted in 23.8 days median survival as compared with 20.8days for vincristine given at its MTD of 1 mg/kg q3d×5 and 13.5 days forvehicle treated mice.

As shown in FIGS. 17 and 18 VBL-NO also demonstrated significantanti-tumor efficacy in the P388 leukemia model. VBL-NO dosed at 30 mg/kgon a q3d×5 schedule resulted in 92% TGD (P<0.001) with a median survivalof 25.9 days as compared to 13.4 days for vehicle treated mice.

Example 15 In Vivo Anti-Tumor Efficacy of Vinca-Alkaloids and AnalogN-Oxides Thereof in Murine Xenograft Models of Human Leukemia and CellsDerived from Hematalogical Malignancies

The in vivo antitumor efficacy of vinca alkaloids and analog N-oxideswere evaluated using murine xenograft models of human hematologicalmalignancies. In one example, female nude mice (n=10) were inoculatedsubcutaneously with 15×10⁶ human K562 myelogenous leukemia cells in a1:1 ratio with matrigel on the right flank. Tumor volumes were monitoredand calculated using the formula: Tumor volume=(a²×b/2) where ‘a’ is thesmallest diameter and ‘b’ is the largest diameter. Once the establishedtumors reached approximately 75-150 mm³ (individual tumor volumes mayrange from 100 to 250 mg) the mice were assigned to the various vehiclecontrol and treatment groups such that the mean tumor volumes in thetreated groups were within 10% of the mean tumor weight in the vehiclecontrol group. On the same day (day 1) and days 4, 7, 10, and 13,vinca-alkaloid N-oxide analogs (VCR-NO, VBL-NO) were administeredintravenously according to a q3d×5 schedule. Mice were also treated withvehicle (negative control) and the standard agents, vinblastine andvincristine (positive controls). Tumor volumes were recorded three timesa week starting when tumors were palpable and including the day of studytermination. Animals were monitored for signs of toxicity and bodyweights were recorded twice a week starting on the first day oftreatment and including the day of study termination. As pre-defined inthe protocol, each animal was actively euthanized if the animal wasfound moribund, if the body weight decreased below 14 g, if theindividual tumor volume reached 3,000 mm³ or if the tumor ulcerated. Themean day of sacrifice were calculated to determine the tumor growthdelay and tumor growth inhibition effects of each test article ascompared to vehicle control. Survival endpoints were defined as a tumorvolume reaching ˜3000 mm³ for each tumor type and Kaplan-Meier plotswere constructed to demonstrate the percentage animals remaining in thestudy as a function of time following treatment. Statisticalsignificance between the treated vs. control groups was evaluated bylogrank analysis.

As indicated in FIG. 19, VCR-NO resulted in a significant dose-dependentK562 tumor growth inhibition. Administered at a 30 mg/kg or 40 mg/kgdose on a q3d×5 schedule, VCR-NO resulted in 60% and 84% tumor growthinhibition (P<0.01) respectively as compared to vehicle control.Similarly, using survival as an endpoint (FIG. 20), VCR-NO dosed at 30mg/kg or 40 mg/kg on a q3d×5 schedule resulted in 48 day and 60 daymedian survival respectively as compared with 30 days for vehicletreated mice. Efficacy seen with VCR-NO was similar to that of thestandard agent vincristine given at its MTD of 1.5 mg/kg q3d×5.

As shown in FIGS. 21 and 22 VBL-NO also demonstrated significantanti-tumor efficacy in the K562 myelogenous leukemia model. VBL-NO dosedat 25 mg/kg or 35 mg/kg on a q3d×5 schedule resulted in 77% and 89%tumor growth inhibition (P<0.01) respectively as compared to vehiclecontrol. Similarly, using survival as an endpoint (FIG. 22), VBL-NOdosed at 25 mg/kg or 35 mg/kg on a q3d×5 schedule resulted in 51 day and57 day median survival respectively as compared with 30 days for vehicletreated mice. Efficacy seen with VBL-NO was similar to that of thestandard agent vinblastine given at its MTD of 2.5 mg/kg q3d×5.

In a related example, the in vivo antitumor efficacy of vinca alkaloidsand analog N-oxides was evaluated using murine xenograft model of humanhematological malignancies, HL60. In this example, female nude mice(n=10) were inoculated subcutaneously with 15×10⁶ human HL60promyelocytic leukemia cells in a 1:1 ratio with matrigel on the rightflank using similar methodology to that described above for K562. Asindicated in FIG. 23, VCR-NO resulted in a significant HL60 tumor growthinhibition. Administered at a 30 mg/kg or 40 mg/kg dose on a q3d×5schedule, VCR-NO resulted in 94% and 96% tumor growth inhibition(P<0.01) respectively as compared to vehicle control. Similarly, usingsurvival as an endpoint (FIG. 24), VCR-NO dosed at 30 mg/kg or 40 mg/kgon a q3d×5 schedule resulted in 60 day and 58 day median survivalrespectively as compared with 21.5 days for vehicle. Efficacy seen withVCR-NO was similar to that of the standard agent vincristine given atits MTD of 1.5 mg/kg q3d×5.

As shown in FIGS. 25 and 26 VBL-NO also demonstrated significantanti-tumor efficacy in the HL60 promyelocytic leukemia model. VBL-NOdosed at 25 mg/kg or 35 mg/kg on a q3d×5 schedule resulted in 91% and94% tumor growth inhibition (P<0.01) respectively as compared to vehiclecontrol. Similarly, using survival as an endpoint (FIG. 26), VBL-NOdosed at 25 mg/kg or 35 mg/kg on a q3d×5 schedule resulted in 60 daymedian survival as compared with 21.5 days for vehicle treated mice.VBL-NO treated mice were still alive at the end of the study and weresacrificed at day 60 according to the protocol.

Example 16 Anti-Tumor Activity of Vinca-Alkaloids and Analog N-OxidesThereof, Alone or in Combination with Chemotherapeutic Agents, in MurineXenograft Models of Human Solid Tumors

The in vivo antitumor efficacy of vinca-alkaloid and analog N-oxides wasevaluated using murine xenograft models of human solid tumors. In thisexample, human HT29 colorectal adenocarcinoma tumor fragments(approximately 1 mm³) were implanted sc into the flank of female nude(nu/nu) mice. When the tumors grew to approximately 80-120 mm in sizethe mice were pair-matched into 10 mice per group. Mice were injected ivwith vinca-alkaloid N-oxide analogs at a range of doses on a q3d×5schedule. Mice were also treated with vehicle (negative control) and thestandard agent, CPT11 (positive control). Tumors were measured withcalipers twice weekly and tumor volume calculated using the formula:Tumor Volume (mm³)=((width)²×length)/2. Animals were monitored for signsof toxicity and weighed daily for the first 5 days of the study and thentwice-weekly until the study end. As pre-defined in the protocol, eachanimal was actively euthanized when its tumor reached the pre-determinedendpoint size of 1000 mm³ or at the conclusion of the study (day 60)whichever came first. The time to endpoint (TTE) was calculated for eachmouse as TTE (days)=(log₁₀(endpoint volume mm³)−b)/m where b is theintercept and m is the slope of the line obtained by linear regressionof a log-transformed tumor growth data set. Treatment outcome wasdetermined from tumor growth delay (TGD) which is defined as theincrease in median TTE in a treatment (T) group as compared to thecontrol (C) group (TGD=T−C) expressed in days or as a percentage of themedian TTE of the control group % TGD=((T−C)/C)×100. Kaplan-Meier plotswere constructed to demonstrate the percentage animals remaining in thestudy as a function of time following treatment. Statisticalsignificance between the treated vs. control groups was evaluated bylogrank analysis.

As indicated in Table 4 and FIG. 27, VCR-NO administered as a singleagent at 15 mg/kg on a q3d×5 schedule resulted in 52% TGD in the HT29colon model. The standard agent, CPT-11 dosed at 100 mg/kg q week×3 alsoresulted in 52% TGD. When VCR-NO and CPT-11 were administered incombination, TGD increased to 111%. As indicated in FIG. 28, efficacywas also demonstrated using survival as an endpoint. As single agents,both VCR-NO dosed at 15 mg/kg q3d×5 and CPT-11 dosed at 100 mg/kg qweek×3 resulted in a median survival of 39.6 days compared to thevehicle control of 26 days. When used in combination VCR-NO dosed at 15mg/kg q3d×5 and CPT-11 dosed at 100 mg/kg q week×3 resulted in a mediansurvival of 55 days. These data provide strong rationale for the use ofthese agents as combination therapy.

Table 4 shows the efficacy of vincristine N-oxide analog (VCR-NO) assingle agent or in combination with CPT-11 in the HT29 colon xenograftmodel in nude mice model (n=10) as determined by Tumor Growth Delay.

VCR-NO VCR-NO VCR-NO (none) (low)* (high)* CPT-11 52% 42% (none) CPT-1171% 59% 74% (low)** CPT-11 52% 111% 82% (high)** *VCR-NO (iv dose): lowdose = 15 mg/kg q3d × 5; high dose = 25 mg/kg q3d × 5 **CPT-11 (ipdose): low dose = 50 mg/kg q week × 3; high dose = 100 mg/kg q week × 3

As indicated in Table 5 and FIG. 29, VBL-NO administered as a singleagent at 20 mg/kg on a q3d×5 schedule resulted in 46% TGD in the HT29colon model. The standard agent, CPT-11 dosed at 50 mg/kg q week×3resulted in 34% TGD. When VBL-NO and CPT-11 were administered incombination, TGD increased to 83%. As indicated in FIG. 30, efficacy wasalso demonstrated using survival as an endpoint. As a single agent,VBL-NO dosed at 20 mg/kg q3d×5 resulted in a median survival of 36.1days, and CPT-11 dosed at 50 mg/kg q week×3 resulted in a mediansurvival of 33.1 days compared to the vehicle control of 24.7 days. Whenused in combination VBL-NO dosed at 20 mg/kg q3d×5 and CPT-11 dosed at50 mg/kg q week×3 resulted in a median survival of 45.1 days. These dataprovide strong rationale for the use of these agents as combinationtherapy.

Table 5 shows the efficacy of vinblastine N-oxide analog (VBL-NO) assingle agent or in combination with CPT-11 in the HT29 colon xenograftmodel in nude mice model (n=10) as determined by Tumor Growth Delay.

VBL-NO VBL-NO VBL-NO (none) (low)* (high)* CPT-11 0% 46% (none) CPT-1134% 58% 83% (low)** CPT-11 60% 54% 82% (high)** *VBL-NO (iv dose): lowdose = 10 mg/kg q3d × 5; high dose = 20 mg/kg q3d × 5 **CPT-11 (ipdose): low dose = 50 mg/kg q week × 3; high dose =100 mg/kg q week × 3.

Having now fully described this invention, it will be understood bythose of ordinary skill in the art that the same can be performed withina wide and equivalent range of conditions, formulations and otherparameters without affecting the scope of the invention or anyembodiment thereof. All patents, patent applications and publicationscited herein are fully incorporated by reference herein in theirentirety.

1. A compound selected from the group consisting of desacetyl vinflunineN-oxide, desacetyl vinorelbine N-oxide, and vinflunine N-oxide, or apharmaceutically acceptable salt thereof.
 2. The compound of claim 1,which is vinflunine N-oxide, or a pharmaceutically acceptable saltthereof.
 3. The compound of claim 2, wherein said pharmaceuticallyacceptable salt is the dihydrochloride salt.
 4. A pharmaceuticalcomposition comprising a compound selected from the group consisting ofvinblastine N-oxide, desacetyl vinblastine N-oxide, vinorelbine N-oxide,vincristine N-oxide, desacetyl vinflunine N-oxide, desacetyl vinorelbineN-oxide, vindesine N-oxide and vinflunine N-oxide or a pharmaceuticallyacceptable salt thereof.
 5. The pharmaceutical composition of claim 4,further comprising a topoisomerase 1 inhibitor.
 6. The pharmaceuticalcomposition of claim 5, wherein said topoisomerase 1 inhibitor isselected from the group consisting of topotecan, irinotecan,9-aminocamptothecin, 10-aminocamptothecin,10,11-methylenedioxycamptothecin and SN-38.
 7. The pharmaceuticalcomposition of claim 4, wherein said compound is vincristine N-oxide, ora pharmaceutically acceptable salt thereof.
 8. The pharmaceuticalcomposition of claim 7, wherein said pharmaceutically acceptable salt isthe dihydrochloride salt.
 9. The pharmaceutical composition of claim 4,wherein said compound is vinblastine N-oxide, or a pharmaceuticallyacceptable salt thereof.
 10. The pharmaceutical composition of claim 9,wherein said pharmaceutically acceptable salt is the dihydrochloridesalt.
 11. The pharmaceutical composition of claim 6, wherein thetopoisomerase 1 inhibitor is irinotecan and the compound is vinblastineN-oxide or vincristine N-oxide.
 12. The pharmaceutical composition ofclaim 4, further comprising one or more active agents independentlyselected from the group consisting of chemotherapeutic agents,anti-angiogenesis agents, vascular targeting agents, HIF1 inhibitors,Hsp90 inhibitors, a tyrosine kinase inhibitor, a serine/threonine kinaseinhibitor, a proteasome inhibitor, an HDAC inibitor, a caspase inducer,a CDK inhibitor, and a proapoptotic molecule.
 13. The pharmaceuticalcomposition of claim 12, wherein said chemotherapeutic agent is selectedfrom the group consisting of abarelix, aldesleukin, alemtuzumab,alitretinoin, allopurinol, altretamine, amifostine, anastrozole, arsenictrioxide, asparaginase, BCG live, bevaceizumab, bexarotene, bleomycin,bortezomib, busulfan, calusterone, camptothecin, capecitabine,carboplatin, carmustine, celecoxib, cetuximab, chlorambucil, cinacalcet,cisplatin, cladribine, cyclophosphamide, cytarabine, dacarbazine,dactinomycin, darbepoetin alfa, daunorubicin, denileukin diftitox,dexrazoxane, docetaxel, doxorubicin, dromostanolone, Elliott's Bsolution, epirubicin, epoetin alfa, estramustine, etoposide, exemestane,filgrastim, floxuridine, fludarabine, fluorouracil, fulvestrant,gemcitabine, gemtuzumab ozogamicin, gefitinib, goserelin, hydroxyurea,ibritumomab tiuxetan, idarubicin, ifosfamide, imatinib, interferonalfa-2a, interferon alfa-2b, irinotecan, letrozole, leucovorin,levamisole, lomustine, meclorethamine, megestrol, melphalan,mercaptopurine, mesna, methotrexate, methoxsalen, methylprednisolone,mitomycin C, mitotane, mitoxantrone, nandrolone, nofetumomab,oblimersen, oprelvekin, oxaliplatin, paclitaxel, pamidronate,pegademase, pegaspargase, pegfilgrastim, pemetrexed, pentostatin,pipobroman, plicamycin, polifeprosan, porfimer, procarbazine,quinacrine, rasburicase, rituximab, sargramostim, streptozocin, talc,tamoxifen, tarceva, temozolomide, teniposide, testolactone, thioguanine,thiotepa, topotecan, toremifene, tositumomab, trastuzumab, tretinoin,uracil mustard, valrubicin, vinblastine, vincristine, vinorelbine, andzoledronate.
 14. The pharmaceutical composition of claim 12, whereinsaid anti-angiogenesis agent is selected from the group consisting ofbevacizumab, angiostatin, endostatin, batimastat, captopril, cartilagederived inhibitor, genistein, interleukin 12, lavendustin,medroxypregesterone acetate, recombinant human platelet factor 4,tecogalan, thrombospondin, TNP-470, anti-VEGF monoclonal antibody,soluble VEGF-receptor chimaeric protein, anti-VEGF receptor antibodies,anti-PDGF receptors, inhibitors of integrins, tyrosine kinaseinhibitors, serine/threonine kinase inhibitors, antisenseoligonucleotides, antisense oligodexoynucleotides, siRNAs, anti-VEGFaptamers and pigment epithelium derived factor.
 15. The compound ofclaim 1, which is desacetyl vinflunine N-oxide, or a pharmaceuticallyacceptable salt thereof.
 16. The compound of claim 1, which is desacetylvinorelbine N-oxide, or a pharmaceutically acceptable salt thereof. 17.The pharmaceutical composition of claim 4, wherein said compound isdesacetyl vinblastine N-oxide, or a pharmaceutically acceptable saltthereof.
 18. The pharmaceutical composition of claim 4, wherein saidcompound is vinorelbine N-oxide, or a pharmaceutically acceptable saltthereof.
 19. The pharmaceutical composition of claim 4, wherein saidcompound is desacetyl vinflunine N-oxide, or a pharmaceuticallyacceptable salt thereof.
 20. The pharmaceutical composition of claim 4,wherein said compound is desacetyl vinorelbine N-oxide, or apharmaceutically acceptable salt thereof.
 21. The pharmaceuticalcomposition of claim 4, wherein said compound is vindesine N-oxide, or apharmaceutically acceptable salt thereof.
 22. The pharmaceuticalcomposition of claim 4, wherein said compound is vinflunine N-oxide, ora pharmaceutically acceptable salt thereof.